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1.
Acta Pharmacol Sin ; 44(12): 2537-2548, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37528233

RESUMO

5-Fluorouracil (5-FU) is the first-line treatment for colorectal cancer (CRC) patients, but the development of acquired resistance to 5-FU remains a big challenge. Deubiquitinases play a key role in the protein degradation pathway, which is involved in cancer development and chemotherapy resistance. In this study, we investigated the effects of targeted inhibition of the proteasomal deubiquitinases USP14 and UCHL5 on the development of CRC and resistance to 5-FU. By analyzing GEO datasets, we found that the mRNA expression levels of USP14 and UCHL5 in CRC tissues were significantly increased, and negatively correlated with the survival of CRC patients. Knockdown of both USP14 and UCHL5 led to increased 5-FU sensitivity in 5-FU-resistant CRC cell lines (RKO-R and HCT-15R), whereas overexpression of USP14 and UCHL5 in 5-FU-sensitive CRC cells decreased 5-FU sensitivity. B-AP15, a specific inhibitor of USP14 and UCHL5, (1-5 µM) dose-dependently inhibited the viability of RKO, RKO-R, HCT-15, and HCT-15R cells. Furthermore, treatment with b-AP15 reduced the malignant phenotype of CRC cells including cell proliferation and migration, and induced cell death in both 5-FU-sensitive and 5-FU-resistant CRC cells by impairing proteasome function and increasing reactive oxygen species (ROS) production. In addition, b-AP15 inhibited the activation of NF-κB pathway, suppressing cell proliferation. In 5-FU-sensitive and 5-FU-resistant CRC xenografts nude mice, administration of b-AP15 (8 mg·kg-1·d-1, intraperitoneal injection) effectively suppressed the growth of both types of tumors. These results demonstrate that USP14 and UCHL5 play an important role in the development of CRC and resistance to 5-FU. Targeting USP14 and UCHL5 with b-AP15 may represent a promising therapeutic strategy for the treatment of CRC.


Assuntos
Neoplasias Colorretais , Complexo de Endopeptidases do Proteassoma , Animais , Camundongos , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Camundongos Nus , Apoptose , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Ubiquitina Tiolesterase
2.
Nucleic Acids Res ; 48(20): 11434-11451, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33080033

RESUMO

Core regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and adolescents carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC 'trio' constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other's super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, CRC-downstream genes and signaling pathways may contain potential therapeutic targets for this malignancy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Metabolismo dos Lipídeos/genética , Camundongos Nus , Proteínas Nucleares , Proteínas de Fusão Oncogênica/fisiologia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Proteína EWS de Ligação a RNA/fisiologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismo , Fatores de Transcrição , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Nucleic Acids Res ; 47(3): 1255-1267, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30496486

RESUMO

As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteína Meis1/genética , Sarcoma de Ewing/genética , Transcrição Gênica , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Humanos , Motivos de Nucleotídeos/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/genética
4.
Proc Natl Acad Sci U S A ; 114(47): 12548-12553, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29109286

RESUMO

The circadian system regulates numerous physiological processes including immune responses. Here, we show that mice deficient of the circadian clock genes Cry1 and Cry2 [Cry double knockout (DKO)] develop an autoimmune phenotype including high serum IgG concentrations, serum antinuclear antibodies, and precipitation of IgG, IgM, and complement 3 in glomeruli and massive infiltration of leukocytes into the lungs and kidneys. Flow cytometry of lymphoid organs revealed decreased pre-B cell numbers and a higher percentage of mature recirculating B cells in the bone marrow, as well as increased numbers of B2 B cells in the peritoneal cavity of Cry DKO mice. The B cell receptor (BCR) proximal signaling pathway plays a critical role in autoimmunity regulation. Activation of Cry DKO splenic B cells elicited markedly enhanced tyrosine phosphorylation of cellular proteins compared with cells from control mice, suggesting that overactivation of the BCR-signaling pathway may contribute to the autoimmunity phenotype in the Cry DKO mice. In addition, the expression of C1q, the deficiency of which contributes to the pathogenesis of systemic lupus erythematosus, was significantly down-regulated in Cry DKO B cells. Our results suggest that B cell development, the BCR-signaling pathway, and C1q expression are regulated by circadian clock CRY proteins and that their dysregulation through loss of CRY contributes to autoimmunity.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/genética , Linfócitos B/imunologia , Relógios Circadianos/imunologia , Criptocromos/imunologia , Animais , Anticorpos Antinucleares/biossíntese , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Relógios Circadianos/genética , Complemento C1q/genética , Criptocromos/deficiência , Criptocromos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Rim/imunologia , Rim/patologia , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/patologia
5.
NAR Cancer ; 6(1): zcad063, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38213995

RESUMO

Cis-regulatory elements (CREs) and super cis-regulatory elements (SCREs) are non-coding DNA regions which influence the transcription of nearby genes and play critical roles in development. Dysregulated CRE and SCRE activities have been reported to alter the expression of oncogenes and tumor suppressors, thereby regulating cancer hallmarks. To address the strong need for a comprehensive catalogue of dysregulated CREs and SCREs in human cancers, we present TSCRE (http://tscre.zsqylab.com/), an open resource providing tumor-specific and cell type-specific CREs and SCREs derived from the re-analysis of publicly available histone modification profiles. Currently, TSCRE contains 1 864 941 dysregulated CREs and 68 253 dysregulated SCREs identified from 1366 human patient samples spanning 17 different cancer types and 9 histone marks. Over 95% of these elements have been validated in public resources. TSCRE offers comprehensive annotations for each element, including associated genes, expression patterns, clinical prognosis, somatic mutations, transcript factor binding sites, cancer-type specificity, and drug response. Additionally, TSCRE integrates pathway and transcript factor enrichment analyses for each study, enabling in-depth functional and mechanistic investigations. Furthermore, TSCRE provides an interactive interface for users to explore any CRE and SCRE of interest. We believe TSCRE will be a highly valuable platform for the community to discover candidate cancer biomarkers.

6.
J Ethnopharmacol ; 301: 115815, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36220508

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Piper longum L., an herbal medicine used in India and other Asian countries, is prescribed routinely for a range of diseases, including tumor. Piperlongumine, a natural product isolated from Piper longum L., has received widespread attention due to its various pharmacological activities, such as anti-inflammatory, antimicrobial, and antitumor effects. AIM OF THE STUDY: Chronic myelogenous leukemia (CML) is a hematopoietic disease caused by Bcr-Abl fusion gene, with an incidence of 15% in adult leukemias. Targeting Bcr-Abl by imatinib provides a successful treatment approach for CML. However, imatinib resistance is an inevitable issue for CML treatment. In particular, T315I mutant is the most stubborn of the Bcr-Abl point mutants associated with imatinib resistance. Therefore, it is urgent to find an alternative approach to conquer imatinib resistance. This study investigated the role of a natural product piperlongumine in overcoming imatinib resistance in CML. MATERIALS AND METHODS: Cell viability and apoptosis were evaluated by MTS assay and Annexin V/propidium iodide counterstaining assay, respectively. Levels of intracellular signaling proteins were assessed by Western blots. Mitochondrial membrane potential was reflected by the fluorescence intensity of rhodamine-123. The function of proteasome was detected using 20S proteasomal activity assay, proteasomal deubiquitinase activity assay, and deubiquitinase active-site-directed labeling. The antitumor effects of piperlongumine were assessed with mice xenografts. RESULTS: We demonstrate that (i) Piperlongumine inhibits proteasome function by targeting 20S proteasomal peptidases and 19S proteasomal deubiquitinases (USP14 and UCHL5) in Bcr-Abl-WT and Bcr-Abl-T315I CML cells; (ii) Piperlongumine inhibits the cell viability of CML cell lines and primary CML cells; (iii) Proteasome inhibition by piperlongumine leads to cell apoptosis and downregulation of Bcr-Abl; (iv) Piperlongumine suppresses the tumor growth of CML xenografts. CONCLUSIONS: These results support that blockade of proteasome activity by piperlongumine provides a new therapeutic strategy for treating imatinib-resistant CML.


Assuntos
Antineoplásicos , Produtos Biológicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Camundongos , Animais , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proliferação de Células , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Fusão bcr-abl/genética , Apoptose , Enzimas Desubiquitinantes/uso terapêutico , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Ubiquitina Tiolesterase/uso terapêutico
7.
Cancer Biol Ther ; 23(1): 319-327, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-35332847

RESUMO

Approximately 40% of patients with diffuse large B-cell lymphoma (DLBCL) are refractory or relapse to standard chemotherapy, and most of them are activated B cell-like DLBCLs (ABC-DLBCL) and germinal center B cell-like DLBCLs (GCB-DLBCL). SNS-032, a novel and selective CDK7/9 inhibitor, that the first phase clinical trials approved by US FDA for cancer treatment have been completed. In this study, we investigated the anti-tumor effect of SNS-032 in ABC- and GCB-DLBCL subtypes. We report that SNS-032 induced growth inhibition and cell apoptosis in both DLBCL cells in vitro, and inhibited the growth of both DLBCL xenografts in nude mice. Mechanistically, SNS-032 inhibited RNA polymerase II, which led to transcriptional-dependent suppression of NF-κB signaling pathway and its downstream targets involved in cell survival; SNS-032 also downregulates BCL-2 and c-MYC in both mRNA and protein levels. Significantly, these findings provide pre-clinical evidence for application of targeting the CDK7/9 in DLBCL.


Assuntos
Linfoma Difuso de Grandes Células B , Recidiva Local de Neoplasia , Animais , Apoptose , Quinases Ciclina-Dependentes , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Nus , Oxazóis , Tiazóis
8.
Clin Transl Med ; 12(9): e1038, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36082692

RESUMO

BACKGROUND: Chronic myeloid leukaemia (CML) is a haematological cancer featured by the presence of BCR-ABL fusion protein with abnormal tyrosine kinase activation. Classical tyrosine kinase inhibitor (TKI)-based therapies are available to patients with CML. However, acquired resistance to TKI has been a challenging obstacle, especially stubborn T315I mutation is the most common cause. Therefore, it is especially urgent to find more effective targets to overcome TKI resistance induced by BCR-ABLT315I . Proteasomal deubiquitinases (USP14 and UCHL5) have fundamental roles in the ubiquitin-proteasome system and possess multiple functions during cancer progression. METHODS: The human peripheral blood mononuclear cells were collected to measure the mRNA expression of USP14 and UCHL5, as well as to detect the toxicity effect of b-AP15. We explored the effect of b-AP15 on the activity of proteasomal deubiquitinases. We detected the effects of b-AP15 on BCR-ABLWT and BCR-ABLT315I CML cells in vitro and in the subcutaneous tumour model. We knocked down USP14 and/or UCHL5 by shRNA to explore whether these proteasomal deubiquitinases are required for cell proliferation of CML. RESULTS: In this study, we found that increased expression of the proteasomal deubiquitinase USP14 and UCHL5 in primary cancer cells from CML patients compared to healthy donors. b-AP15, an inhibitor of USP14 and UCHL5, exhibited potent tumour-killing activity in BCR-ABLWT and BCR-ABLT315I CML cell lines, as well as in CML xenografts and primary CML cells. Mechanically, pharmacological or genetic inhibition of USP14 and UCHL5 induced cell apoptosis and decreased the protein level of BCR-ABL in CML cells expressing BCR-ABLWT and BCR-ABLT315I . Moreover, b-AP15 synergistically enhanced the cytotoxic effect caused by TKI imatinib in BCR-ABLWT and BCR-ABLT315I CML cells. CONCLUSION: Collectively, our results demonstrate targeting USP14 and UCHL5 as a potential strategy for combating TKI resistance in CML.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteínas Quinases , Ubiquitina Tiolesterase , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Fusão bcr-abl/farmacologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Piperidonas/metabolismo , Piperidonas/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética
9.
Colloids Surf B Biointerfaces ; 208: 112100, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34547704

RESUMO

The adverse effect and drug resistance of Cisplatin (CDDP) could be potential reduced by delivering in targeted nanoparticles and by combining with adjuvant therapy such as photodynamic therapy. In this study, F/CDPR-NP was formulated and characterized for all the physicochemical, biological and in vivo analysis. The results obtained from various in vitro and biological studies showed that encapsulation of CDDP and PBR in PLGA nanoparticles results in controlled release of encapsulated drugs and exhibited significantly low cell viability in CNE-1 and HNE-1 cancer cells. F/CDPR-NP significantly prolonged the blood circulation of the encapsulated drugs. The AUC of CDDP from F/CDPR-NP (4-fold) was significantly higher compared to that of free CDDP and similarly significantly higher t1/2 for CDDP from F/CDPR-NP was observed. F/CDPR-NP in the presence of laser irradiation showed significant reduction in the tumor burden with low tumor cell proliferations compared to either CDPR-NP or free CDDP indicating the potential of targeted nanoparticles and photodynamic therapy. Overall, combination of treatment modalities and active targeting approach paved way for the higher antitumor activity in nasopharyngeal carcinoma model. The positive results from this study will show new horizon for the treatment of other cancer models.


Assuntos
Antineoplásicos , Nanopartículas , Neoplasias Nasofaríngeas , Fotoquimioterapia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ácido Fólico , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico
10.
Bioorg Med Chem ; 18(5): 1806-15, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20149665

RESUMO

Imatinib (STI571) is the frontline targeted-therapeutic agent for patients with chronic myelogenous leukemia (CML). However, resistance to imatinib due to point mutations in Bcr-Abl kinase domain is an emerging problem. We recently reported that triptolide (compound 1) could effectively kill CML cells including those harboring T315I mutant Bcr-Abl. In the present study, we designed a series of C-14 triptolide derivatives with C-14-hydroxyl substituted by different amine esters (3-18): 3-6 and 13 (by aliphatic chain amine esters); 7-9, 11, 12 and 15-18 (by alicyclic amine esters with different size), and 10 and 14 (by aralkylamine esters).The compounds were examined for their antineoplastic activity against CML cells (including KBM5-T315I cells) in terms of proliferation inhibition, apoptosis and signal transduction. Nude mouse xenograft model was also used to evaluate the in vivo activity. Compounds 2-9, 11-14, 17 and 18 exhibited a potent inhibitory activity against KBM5 and KBM5-T315I cells. This series of derivatives down-regulated Bcr-Abl mRNA. Compounds 4, 5, 8 and 9 were further examined for their impact on signaling and apoptosis with immunoblotting. Compound 5 was chosen for evaluation in a nude mouse xenograft model. The stereo-hindrance of C-14 group appeared to be responsible for the antitumor effect. The computational small molecule-protein docking analysis illustrated the possible interaction between compound 9 and RNA polymerase II. Our results suggest that this series of derivatives may be promising agents to overcome imatinib-resistance caused by the Bcr-Abl-T315I mutation.


Assuntos
Antineoplásicos/síntese química , Diterpenos/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Fenantrenos/química , Substituição de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose , Benzamidas , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Diterpenos/síntese química , Diterpenos/uso terapêutico , Regulação para Baixo , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Compostos de Epóxi/síntese química , Compostos de Epóxi/química , Compostos de Epóxi/uso terapêutico , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Camundongos , Camundongos Nus , Fenantrenos/síntese química , Fenantrenos/uso terapêutico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , RNA Mensageiro/metabolismo , Transdução de Sinais , Água/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Clin Cancer Res ; 15(5): 1686-97, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19240172

RESUMO

PURPOSE: Resistance to STI571 is an emerging problem for patients with chronic myelogenous leukemia (CML). Mutation in the kinase domain of Bcr-Abl is the predominant mechanism of the acquired resistance to STI571. In the present study, we investigated the effect of triptolide on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. EXPERIMENTAL DESIGN: CML cell lines (KBM5 versus KBM5-T315I, BaF3-Bcr-Abl versus BaF3-Bcr-Abl-T315I) and primary cells from CML patients with clinical resistance to STI571 were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. Nude mouse xenograft model was also used to evaluate the antitumor activity. RESULTS: Triptolide potently down-regulated the mRNA and protein levels of Bcr-Abl independently of the caspase or proteosome activation in CML cells. It induced mitochondrial-dependent apoptosis in Bcr-Abl-T315I CML cells and primary cells from CML patients with clinical resistance to STI571. Additionally, triptolide inhibited the growth of STI571-sensitive KBM5 and STI571-resistant KBM5-T315I CML cells in nude mouse xenografts. Triptolide also down-regulated the expression of survivin, Mcl-1, and Akt in CML cells, which suggests that it may have multiple targets. CONCLUSIONS: These findings suggest that triptolide is a promising agent to overcome STI571-resistant CML cells, and warrant a clinical trial of triptolide derivatives for CML with Bcr-Abl-T315I mutation.


Assuntos
Apoptose/efeitos dos fármacos , Diterpenos/uso terapêutico , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Mutação/genética , Fenantrenos/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Antineoplásicos Alquilantes/uso terapêutico , Benzamidas , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Compostos de Epóxi/uso terapêutico , Feminino , Citometria de Fluxo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Técnicas Imunoenzimáticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirimidinas/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Tripterygium/química , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
12.
Eur J Pharmacol ; 876: 173064, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179085

RESUMO

The selective BCR-ABL tyrosine kinase inhibitor imatinib is one of the first-line therapies in the management of chronic myeloid leukaemia (CML). However, acquired resistance to this inhibitor, which is especially conferred by the T315I point mutation in BCR-ABL, impedes the efficacy of imatinib therapy. Therefore, the discovery and development of novel agents to overcome imatinib resistance is urgently needed. Pseudolaric acid B (PAB), a small molecule isolated from the traditional Chinese medicine Cortex pseudolaricis, has been reported to be a potential candidate for immune disorders and cancer treatment. However, its effects on CML and the involved molecular mechanism have not been reported. In the current study, by performing both in vitro and in vivo experiments in CML cells, we showed that PAB blocked the cell cycle at G2/M phase and subsequently activated the caspase pathway, cleaved the BCR-ABL protein and inhibited the BCR-ABL downstream pathways, ultimately leading to cell proliferation inhibition, cytotoxicity and apoptosis. These events were observed in both imatinib-sensitive and imatinib-insensitive CML cell lines. Moreover, PAB decreased the viability of primary blood mononuclear cells from CML patients and induced apoptosis in these cells. Our findings suggest that PAB could be used as a novel agent to sensitize imatinib-resistant CML.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Mitose/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Rep ; 10(1): 1216, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988304

RESUMO

Advanced and therapy-resistant prostate tumors often display neural or neuroendocrine behavior. We assessed the consequences of prostate cancer cell interaction with neural cells, which are rich in the human prostate and resident of the prostate tumor. In 3-dimensional co-culture with neurospheres, red fluorescent human LNCaP cells formed agglomerates on the neurosphere surface. Upon induced neural differentiation, some red fluorescent cells showed morphology of fully differentiated neural cells, indicating fusion between the cancer and neural stem cells. These fusion hybrids survived for extended times in a quiescent state. A few eventually restarted cell division and propagated to form derivative hybrid progenies. Clones of the hybrid progenies were highly heterogeneous; most had lost prostatic and epithelial markers while some had acquired neural marker expression. These results indicate that cancer cells can fuse with bystander neural cells in the tumor microenvironment; and cancer cell fusion is a direct route to tumor cell heterogeneity.


Assuntos
Células-Tronco Neurais/metabolismo , Células Neuroendócrinas/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Fusão Celular/métodos , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Técnicas de Cocultura/métodos , Humanos , Masculino , Células-Tronco Neurais/fisiologia , Sistemas Neurossecretores/citologia , Próstata/citologia , Neoplasias da Próstata/imunologia , Ratos , Células Estromais/citologia , Microambiente Tumoral/fisiologia
14.
Cancer Sci ; 100(7): 1335-43, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19383029

RESUMO

Gain-of-function mutations of the receptor tyrosine kinase KIT can cause systemic mastocytosis (SM) and gastrointestinal stromal tumors. Most of the constitutively active KIT can be inhibited by imatinib; D816V KIT cannot. In this study, we investigated the activity of triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook. f., in cells expressing mutant KIT, including D816V KIT. Imatinib-sensitive HMC-1.1 cells harboring the mutation V560G in the juxtamembrane domain of KIT, imatinib-resistant HMC-1.2 cells harboring both V560G and D816V mutations, and murine P815 cells, were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. The in vivo antitumor activity was evaluated by using the nude mouse xenograft model. Our results demonstrated that triptolide potently inhibits the growth of both human and murine mast cells harboring not only imatinib-sensitive KIT mutation but also imatinib-resistant D816V KIT. Triptolide markedly inhibited KIT mRNA levels and strikingly reduced the levels of phosphorylated and total Stat3, Akt, and Erk1/2, downstream targets of KIT. Triptolide triggered apoptosis by inducing depolarization of mitochondrial potential and release of cytochrome c, downregulation of Mcl-1 and XIAP. Furthermore, triptolide significantly abrogated the growth of imatinib-resistant HMC-1.2 cell xenografts in nude mice and decreased KIT expression in xenografts. Our data demonstrate that triptolide inhibits imatinib-resistant mast cells harboring D816V KIT. Further investigation of triptolide for treatment of human neoplasms driven by gain-of-function KIT mutations is warranted.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Diterpenos/farmacologia , Mastócitos/efeitos dos fármacos , Mutação , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Regulação para Baixo , Compostos de Epóxi/farmacologia , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/metabolismo
15.
J Exp Clin Cancer Res ; 38(1): 453, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694672

RESUMO

BACKGROUND: The first line therapy for patients with diffuse large B cell (DLBCL) is R-CHOP. About half of DLBCL patients are either refractory to, or will relapse, after the treatment. Therefore, identifying novel drug targets and effective therapeutic agents is urgently needed for improving DLBCL patient survival. b-AP15, a selective small molecule inhibitor of proteasomal USP14 and UCHL5 deubiquitinases (DUBs), has shown selectivity and efficacy in several other types of cancer cells. This is the first study to report the effect of b-AP15 in DLBCL. METHODS: Cell lines of two DLBCL subtypes, Germinal Center B Cell/ GCB (SU-DHL-4, OCI-LY-1, OCI-LY-19) and Activated B Cell/ABC (SU-DHL-2), were used in the current study. Cell viability was measured by MTS assay, proliferation by trypan blue exclusion staining assay, cellular apoptosis by Annexin V-FITC/PI staining and mitochondrial outer membrane permeability assays, the activities of 20S proteasome peptidases by cleavage of specific fluorogenic substrates, and cell migration was detected by transwell assay in these GCB- and ABC-DLBCL cell lines. Mouse xenograft models of SU-DHL-4 and SU-DHL-2 cells were used to determine in vivo effects of b-AP15 in DLBCL tumors. RESULTS: b-AP15 inhibited proteasome DUB activities and activated cell death pathway, as evident by caspase activation and mitochondria apoptosis in GCB- and ABC- DLBCL cell lines. b-AP15 treatment suppressed migration of GCB- and ABC-DLBCL cells via inhibiting Wnt/ß-catenin and TGFß/Smad pathways. Additionally, b-AP15 significantly inhibited the growth of GCB- and ABC DLBCL in xenograft models. CONCLUSIONS: These results indicate that b-AP15 inhibits cell migration and induces apoptosis in GCB- and ABC-DLBCL cells, and suggest that inhibition of 19S proteasomal DUB should be a novel strategy for DLBCL treatment.


Assuntos
Apoptose/efeitos dos fármacos , Enzimas Desubiquitinantes/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/metabolismo , Piperidonas/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cell Death Dis ; 8(5): e2803, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28518145

RESUMO

Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cisplatino/uso terapêutico , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Deleção de Genes , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Gradação de Tumores , Complexo Shelterina , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Telômeros
18.
Cell Death Dis ; 8(7): e2913, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28682311

RESUMO

Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. T315I Bcr-Abl is the most notorious point mutation to elicit acquired resistance to imatinib (IM), leading to poor prognosis. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that platinum pyrithione (PtPT) potently inhibits the ubiquitin-proteasome system (UPS) via targeting the 26 S proteasome-associated deubiquitinases (DUBs), without effecting on the 20 S proteasome. Here we further report that (i) PtPT induces apoptosis in Bcr-Abl wild-type and Bcr-Abl-T315I mutation cells including the primary mononuclear cells from CML patients clinically resistant to IM, as well as inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) PtPT downregulates Bcr-Abl level through restraining Bcr-Abl transcription, and decreasing Bcr-Abl protein mediated by DUBs inhibition-induced caspase activation; (iii) UPS inhibition is required for PtPT-induced caspase activation and cell apoptosis. These findings support that PtPT overcomes IM resistance through both Bcr-Abl-dependent and -independent mechanisms. We conclude that PtPT can be a lead compound for further drug development to overcome imatinib resistance in CML patients.


Assuntos
Antineoplásicos/farmacologia , Caspases/genética , Enzimas Desubiquitinantes/genética , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Bortezomib/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Oncotarget ; 8(8): 13942-13956, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28086217

RESUMO

The ubiquitin-proteasome system (UPS) plays a central role in various cellular processes through selectively degrading proteins involved in critical cellular functions. Targeting UPS has been validated as a novel strategy for treating human cancer, as inhibitors of the 20S proteasome catalytic activity are currently in clinical use for treatment of multiple myeloma and other cancers, and the deubiquitinase activity associated with the proteasome is also a valid target for anticancer agents. Recent studies suggested that zinc pyrithione, an FDA-approved antidandruff agent, may have antitumor activity, but the detailed molecular mechanisms remain unclear. Here we report that zinc pyrithione (ZnPT) targets the proteasome-associated DUBs (USP14 and UCHL5) and inhibits their activities, resulting in a rapid accumulation of protein-ubiquitin conjugates, but without inhibiting the proteolytic activities of 20S proteasomes. Furthermore, ZnPT exhibits cytotoxic effects against various cancer cell lines in vitro, selectively kills bone marrow cells from leukemia patients ex vivo, and efficiently inhibits the growth of lung adenocarcinoma cancer cell xenografts in nude mice. This study has identified zinc pyrithione, an FDA-approved pharmacological agent with potential antitumor properties as a proteasomal DUB inhibitor.


Assuntos
Antineoplásicos/farmacologia , Ceratolíticos/farmacologia , Neoplasias Experimentais/patologia , Compostos Organometálicos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Caspa/tratamento farmacológico , Imunofluorescência , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Modelos Moleculares , Inibidores de Proteassoma/farmacologia , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cell Death Dis ; 8(6): e2877, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28617443

RESUMO

The molecular mechanism underlying bilirubin neurotoxicity remains obscure. Ubiquitin-proteasome system-mediated proteolysis is pivotal to virtually all cellular processes and cell survival. Here we report for the first time that bilirubin at a clinically relevant elevated level impairs proteasomal function via inhibiting both the 19S proteasome-associated deubiquitinases (USP14 and UCHL5) and the chymotrypsin-like (CT-like) peptidase activity of 20S proteasomes, thereby contributing to bilirubin neurotoxicity. This is supported by multiple lines of evidence. First, sera from patients with hyperbilirubinemia were able to inhibit the peptidase activity of purified 20S proteasome in vitro in a bilirubin concentration-dependent manner; meanwhile, the blood cells of these patients showed significantly increased levels of ubiquitinated proteins (Ub-prs), consistent with proteasome inhibition. Second, intracerebroventricular injection to adult rats or intraperitoneal injections to neonatal rats of bilirubin-induced neural accumulation of Ub-prs, concurrent with other neural pathology; and brain malfunction and pathology induced by neonatal exposure to hyperbilirubinemia were detectable in the rats during their adulthood. Third, in primary cultures of hippocampal neurons, bilirubin strikingly induced Ub-pr accumulation before the activation of cell death pathway becomes discernible. Finally, bilirubin in vitro directly inhibited both the deubiquitination activity of proteasome-associated USP14 and UCHL5 and the CT-like peptidase activity of purified 20S proteasomes, in a dose-dependent manner. Hence, this study has discovered that increased bilirubin at a clinically achievable level can act as a proteasome inhibitor via targeting the 19S proteasome-associated deubiquitinases (DUBs) and, perhaps to a less extent, the 20S proteasome, identifying a novel mechanism for bilirubin neurotoxicity.


Assuntos
Bilirrubina/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Adulto , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Quimotripsina/química , Cognição/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hipocampo/metabolismo , Humanos , Masculino , Aprendizagem em Labirinto , Memória , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Neurônios/metabolismo , Proteólise , Ratos , Aprendizagem Espacial , Ubiquitinação/efeitos dos fármacos
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