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1.
PLoS One ; 15(1): e0223463, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914134

RESUMO

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.


Assuntos
Citidina Desaminase/genética , Dano ao DNA/genética , Antígenos de Histocompatibilidade Menor/genética , Mieloma Múltiplo/genética , Anticorpos Monoclonais Humanizados/farmacologia , Bortezomib/farmacologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/radioterapia , Mutação/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Ácidos Polimetacrílicos/farmacologia , RNA Interferente Pequeno/genética , Radiação , Transdução de Sinais/efeitos dos fármacos
2.
Int J Cancer ; 146(7): 1963-1978, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390487

RESUMO

E3 ubiquitin ligases primarily determine the substrate specificity of the ubiquitin-proteasome system and play an essential role in the resistance to bortezomib in multiple myeloma (MM). Neural precursor cell-expressed developmentally downregulated gene 4-1 (NEDD4-1, also known as NEDD4) is a founding member of the NEDD4 family of E3 ligases and is involved in the proliferation, migration, invasion and drug sensitivity of cancer cells. In the present study, we investigated the role of NEDD4-1 in MM cells and explored its underlying mechanism. Clinically, low NEDD4-1 expression has been linked to poor prognosis in patients with MM. Functionally, NEDD4-1 knockdown (KD) resulted in bortezomib resistance in MM cells in vitro and in vivo. The overexpression (OE) of NEDD4-1, but not an enzyme-dead NEDD4-1-C867S mutant, had the opposite effect. Furthermore, the overexpression of NEDD4-1 in NEDD4-1 KD cells resensitized the cells to bortezomib in an add-back rescue experiment. Mechanistically, pAkt-Ser473 levels and Akt signaling were elevated and decreased by NEDD4-1 KD and OE, respectively. NEDD4-1 ubiquitinated Akt and targeted pAkt-Ser473 for proteasomal degradation. More importantly, the NEDD4-1 KD-induced upregulation of Akt expression sensitized MM cells to growth inhibition after treatment with an Akt inhibitor. Collectively, our results suggest that high NEDD4-1 levels may be a potential new therapeutic target in MM.


Assuntos
Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/patologia , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Animais , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Ubiquitina-Proteína Ligases Nedd4/genética , Cultura Primária de Células , Prognóstico , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Eur J Med Chem ; 186: 111906, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31787362

RESUMO

Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors.


Assuntos
Bortezomib/farmacologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Inibidores de Proteassoma/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Bortezomib/síntese química , Bortezomib/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Estrutura Molecular , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Relação Estrutura-Atividade
4.
Anticancer Res ; 39(12): 6673-6684, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810932

RESUMO

BACKGROUND/AIM: The aim of the study was to evaluate the antitumor potential and combination effects of chemotherapeutic drugs. MATERIALS AND METHODS: The cytotoxicity of 20 drip-type classical and molecular-targeted anticancer drugs was examined against 4 human oral squamous cell carcinoma cell lines and 5 human oral normal mesenchymal and epithelial cells. Cell cycle progression was monitored by a cell sorter. Combination effect was evaluated by combination index. RESULTS: Most of the classical anticancer drugs showed much higher antitumor activity than molecular-targeted drugs, except bortezomib. Among 12 classical anticancer drugs, taxanes and gemsitabine showed the highest tumor-specificity (TS) and potency-selectivity expression (PSE) values, whereas platinum analogs showed the least TS value. Combination of two classical or a classical and a molecular-targeted drug showed mostly additive or antagonistic effect. 5-FU and cisplatin did not produce a subG1 population, but induced G2/M or G1/S arrest, regardless of the addition of cetuximab. Cetuximab, nibolumab and bortezomib showed potent keratinocyte toxicity. CONCLUSION: The present TS monitoring system may provide useful information for building up the treatment regimens of anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias Bucais/tratamento farmacológico , Bortezomib/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cetuximab/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Fluoruracila/farmacologia , Hormese , Humanos , Técnicas In Vitro , Queratinócitos/efeitos dos fármacos , Mucosa Bucal/citologia , Nivolumabe/farmacologia , Compostos de Platina/farmacologia , Taxoides/farmacologia
5.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569723

RESUMO

Although dual EGFR/HER2 tyrosine kinase inhibitor lapatinib has provided effective clinical benefits for HER2-positive breast cancer patients, acquired resistance to this drug remains a major concern. Thus, the development of alternative therapeutic strategies is urgently needed for patients who failed lapatinib treatment. Proteasome inhibitors have been reported to possess high anti-tumor activity to breast cancer cells. Therefore, this study aims to examine whether and how proteasome inhibitor bortezomib can overcome lapatinib resistance. Treatments with several proteasome inhibitors, including Bortezomib, MG132, and proteasome inhibitor I (PSI), as well as the viabilities of both HER2-positive breast cancer cell lines and their lapatinib-resistant clones, were inhibited. Importantly, the expressions of ErbB family were downregulated at both transcriptional and translational levels. Also, our results further indicated that proteasome inhibitors decreased ErbB family expression through lysosomal degradation pathway in a heat shock protein 90 (HSP90)-dependent manner. In this study, our data supported a potential approach to overcome the acquired resistance of HER2-overexpressing breast cancer patients to lapatinib using proteasome inhibitors.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Lisossomos/metabolismo , Inibidores de Proteassoma/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteólise , Transdução de Sinais
6.
Virol Sin ; 34(5): 572-582, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31637631

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptide-boronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded non-structural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.


Assuntos
Antivirais/farmacologia , Bortezomib/farmacologia , Phlebovirus/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Apoptose , Células HEK293 , Humanos , Transdução de Sinais , Carga Viral , Replicação Viral/efeitos dos fármacos
7.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 311-316, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631595

RESUMO

Objective: To investigate the anti-tumor effect of bortezomib on extranodal natural killer/T cell lymphoma, nasal type (ENKTL). Methods: SNK-6 cells were treated with different mass concentrations of bortezomib (0, 1, 2, 4, 5, 6 ng/mL) for 24, 48, 72 h, and different concentrations of nuclear factor-kappa B (NF-κB) signaling pathway inhibitor BAY11-7082 (0, 1, 2, 2.5, 5, 10, 20 µmol/L) for 24 h respectively, then the cell viability was measured by CCK8 kit and the half inhibitory concentration (IC 50) was calculated. SNK-6 cells were treated with 30µmol/L Z-VAD-FMK (Pan-caspase inhibitor)+3ng/mL bortezomib, and 5, 10 µmol/L BAY11-7082+3 ng/mL bortezomib for 24 h respectively, then the cell viability was measured by CCK8 kit. After treatment of SNK-6 cells with different mass concentrations of bortezomib for 24 h, apoptosis was detected by AnnexinⅤ/PI flow cytometry; the expression of apoptosis-related protein Caspase-3, poly ADP-ribose polymerase (PARP) and Bcl-2 and NF-κB signaling pathway key proteins P65 and P100/P52 were detected by Western blot. Results: Bortezomib inhibited the proliferation of SNK-6 cells in a dose-dependent manner ( P<0.05), and IC 50( (2.87±0.06) ng/mL) at 24 h was lower than that at 48 h and 72 h ( P<0.05). BAY11-7082 also inhibited the proliferation of SNK-6 cells with an IC 50= (9.73±0.36) µmol/L at 24 h. The combination treatment indicated that Z-VAD-FMK could attenuate the inhibitory effect of bortezomib on the proliferation of SNK-6 cells ( P<0.05), while BAY11-7082 could enhance the inhibitory effect of bortezomib on the proliferation of SNK-6 cells ( P<0.05). After treatment of SNK-6 cells with bortezomib for 24 h, apoptosis-related protein Caspase-3 cleavage, PARP activation, and Bcl-2 cleavage; NF-κB signaling pathway-related protein P65 phosphorylation level decreased, and P52 decreased. Conclusion: Bortezomib inhibits ENKTL cells proliferation by inhibiting NF-κB signaling pathway and induces apoptosis of ENKTL cells via mitochondria-mediated caspase pathway.


Assuntos
Apoptose , Bortezomib/farmacologia , Linfoma Extranodal de Células T-NK/patologia , NF-kappa B , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Nitrilos/farmacologia , Sulfonas/farmacologia
8.
BMC Cancer ; 19(1): 975, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638931

RESUMO

BACKGROUND: Multiple myeloma (MM) cells gain protection against drugs through interaction with bone marrow stromal cells (BMSCs). This form of resistance largely accounts for resistance to therapy in MM patients which warrants further exploration to identify more potential therapeutic targets. METHODS: We performed miRNA/mRNA qPCR arrays and western blotting to analyze transcriptional and translational changes in MM cells co-cultured with BMSCs. Drug cytotoxicity and apoptosis in MMGFP-BMSC co-cultures were measured using fluorescence plate reader and flowcytometry, respectively. miRNA was overexpressed in MM cell lines using Lentiviral transduction, miRNA-3'UTR binding was examined using luciferase assay. RESULTS: We found that BMSCs downregulated miR-101-3p and upregulated survivin (BIRC5) in MM cells. Survivin was downregulated by miR-101-3p overexpression and found to be a direct target of miR-101-3p using 3'UTR luciferase assay. Overexpression of survivin increased viability of MM cells in the presence of anti-myeloma drugs, and miR-101-3p inhibition by anti-miR against miR-101-3p upregulated survivin. Furthermore, overexpression of miR-101-3p or silencing of survivin triggered apoptosis in MM cells and sensitized them to anti-myeloma drugs in the presence of BMSCs overcoming the stroma-induced drug resistance. CONCLUSIONS: Our study demonstrates that BMSC-induced resistance to drugs is associated with survivin upregulation which is a direct target of miR-101-3p. This study also identifies miR-101-3p-survivin interaction as a druggable target involved in stroma-mediated drug resistance in MM and suggests it for developing more efficient therapeutic strategies.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Expressão Ectópica do Gene/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Survivina/genética , Regiões 3' não Traduzidas/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MicroRNAs/antagonistas & inibidores , Mieloma Múltiplo/patologia , Transfecção
9.
Eur J Med Chem ; 182: 111646, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31521028

RESUMO

The immunoproteasome, a specialized form of proteasome, is mainly expressed in lymphocytes and monocytes of jawed vertebrates and responsible for the generation of antigenic peptides for cell-mediated immunity. Overexpression of immunoproteasome have been detected in a wide range of diseases including malignancies, autoimmune and inflammatory diseases. Following the successful approval of constitutive proteasome inhibitors bortezomib, carfilzomib and Ixazomib, and with the clarification of immunoproteasome crystal structure and functions, a variety of immunoproteasome inhibitors were discovered or rationally developed. Not only the inhibitory activities, the selectivities for immunoproteasome over constitutive proteasome are essential for the clinical potential of these analogues, which has been validated by the clinical evaluation of immunoproteasome-selective inhibitor KZR-616 for the treatment of systemic lupus erythematosus. In this review, structure, function as well as the current developments of various inhibitors against immunoproteasome are going to be summarized, which help to fully understand the target for drug discovery.


Assuntos
Antineoplásicos/farmacologia , Doenças Autoimunes/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/química , Doenças Autoimunes/metabolismo , Compostos de Boro/química , Compostos de Boro/farmacologia , Bortezomib/química , Bortezomib/farmacologia , Glicina/análogos & derivados , Glicina/química , Glicina/farmacologia , Neoplasias Hematológicas/metabolismo , Humanos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/química
10.
J Neurooncol ; 144(3): 463-473, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31392596

RESUMO

INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal form of gliomas. New therapies are currently in development to tackle treatment limitations such as chemotherapy resistance. One mechanism of resistance may be the stress granules (SG) assembly, a stress-related cellular response that allows cells to recruit and protect mRNAs during stress. SG are composed of various proteins, being G3BP1 a core element that enucleates and results in SG assembly. Here, we aimed to evaluate the effects of inhibiting the G3PB1 expression in the chemotherapeutical-induced cell death of the U87 glioblastoma cell line. MATERIALS AND METHODS: G3BP1 mRNA and protein expression were modulated with short-interference RNA (siRNA). The viability of U87 cells after Bortezomib (BZM), a proteasome inhibitor, and Temozolomide (TMZ), an alkylating agent, was assessed by MTT assay. Apoptosis was evaluated by staining cells with Annexin-V/7-AAD and analyzing by flow cytometry. Caspase-3 activation was evaluated by immunoblotting. The chorioallantoic membrane in vivo assay was used to evaluate angiogenesis. RESULTS: When G3BP1 was knocked-down, the SG assembly was reduced and the BZM-treated cells, but not TMZ-treated cells, had a significant increase in the apoptotic response. Corroborating this data, we observed increased Caspase-3 activation in the BZM-treated and G3BP1-knocked-down cells when compared to vehicle-treated and scramble-transfected cells. Worth mentioning, the conditioned culture medium of G3BP1-knocked-down BZM-treated cells inhibited angiogenesis when compared to controls. CONCLUSION: Our data suggest G3BP1 knockdown diminishes SG formation and stimulates BZM-induced apoptosis of U87 cells in vitro, in addition to inhibiting glioblastoma-induced angiogenesis in vivo.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , DNA Helicases/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Antineoplásicos Alquilantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Grânulos Citoplasmáticos/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Temozolomida/farmacologia , Células Tumorais Cultivadas
11.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
12.
Oncogene ; 38(41): 6737-6751, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31406244

RESUMO

MYCN amplification in neuroblastoma predicts poor prognosis and resistance to therapy. Yet pharmacological strategies of direct MYC inhibition remain unsuccessful due to its "undruggable" protein structure. We herein developed a synthetic lethal screen against MYCN-amplified neuroblastomas using clinically approved therapeutic reagents. We performed a high-throughput screen, from a library of 938 FDA-approved drugs, for candidates that elicit synthetic lethal effects in MYC-driven neuroblastoma cells. The proteasome inhibitors, which are FDA approved for the first-line treatment of multiple myeloma, emerge as top hits to elicit MYC-mediated synthetic lethality. Proteasome inhibition activates the PERK-eIF2α-ATF4 axis in MYC-transformed cells and induces BAX-mediated apoptosis through ATF4-dependent NOXA and TRIB3 induction. A combination screen reveals the proteasome inhibitor bortezomib (BTZ) and the histone deacetylase (HDAC) inhibitor vorinostat (SAHA) concertedly induce dramatic cell death in part through synergistic activation of BAX. This combination causes marked tumor suppression in vivo, supporting dual proteasome/HDAC inhibition as a potential therapeutic approach for MYC-driven cancers. This FDA-approved drug screen with in vivo validation thus provides a rationale for clinical evaluation of bortezomib, alone or in combination with vorinostat, in MYC-driven neuroblastoma patients.


Assuntos
Antineoplásicos/farmacologia , Genes myc , Neuroblastoma/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Autoantígeno Ku/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Estados Unidos , United States Food and Drug Administration , Vorinostat/farmacologia , Proteína X Associada a bcl-2/metabolismo , eIF-2 Quinase/metabolismo
13.
Strahlenther Onkol ; 195(10): 934-939, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363801

RESUMO

PURPOSE: The urinary bladder is one major organ at risk in radiotherapy of pelvic malignancies. The radiation response manifests in early and chronic changes in bladder function. These are based on inflammatory effects and changes in urothelial cell function and proliferation. This study evaluates the effect of bortezomib as an anti-proliferative and anti-inflammatory compound in an established mouse bladder model. The early radiation-induced bladder dysfunction in the mouse occurs in two phases during the first month after irradiation (phase I: day 0-15, phase II: days 16-30). MATERIALS AND METHODS: Daily bortezomib injections (0.02 mg/ml, subcutaneously) were administered between days 0-15 or 15-30 in separate groups. Single graded radiation doses were administered in five dose groups. Cystometry was carried out before (individual control) and during the first month after irradiation. When bladder capacity was decreased by ≥50%, mice were considered as responders. Statistical analysis was performed by the SPSS software version 24. RESULTS: Daily bortezomib injections between days 0-15 resulted in a significant decrease in responders for phase I. There was no significant effect with daily bortezomib injections between days 16-30. CONCLUSION: Two separate waves of acute radiation-induced urinary bladder dysfunction have distinct mechanisms that need further biological studies.


Assuntos
Bortezomib/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Bexiga Urinária/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Esquema de Medicação , Feminino , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C3H , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/efeitos da radiação , Órgãos em Risco/efeitos da radiação , Bexiga Urinária/efeitos dos fármacos , Urodinâmica/efeitos dos fármacos , Urodinâmica/efeitos da radiação
14.
Int Immunopharmacol ; 75: 105758, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377589

RESUMO

OBJECTIVE: The present study aimed to investigate the functional role of bortezomib in the development of acute allograft rejection (AR) after renal transplant. METHODS: The mouse model of AR was established by allograft kidney transplant followed by the treatment of bortezomib. The serum cytokines, renal function, and the percentage of T follicular helper (Tfh) cells in CD4+ T cells were measured. The effect of miR-15b and interferon-regulatory factor 4 (IRF4) on Tfh cell proliferation and differentiation was assessed by cell transfection technology and CCK-8 assay. The interaction between miR-15b and IRF4 was assessed by luciferase reporter assay. RESULTS: Bortezomib relieved acute AR after renal transplant by suppressing Tfh cell proliferation and differentiation. Meanwhile, bortezomib treatment markedly increased miR-15b expression in AR renal tissues. The upregulation of miR-15b inhibited Tfh cell proliferation and differentiation by reducing IRF4. In addition, bortezomib ameliorated AR by suppressing Tfh cell proliferation and differentiation through miR-15b/IRF4 axis in vitro and in vivo. CONCLUSION: Our findings indicated the mechanism underlying the bortezomib in treating acute AR after renal transplant, and suggested the critical role of miR-15b in Tfh cell proliferation and differentiation, which provided a therapeutic target in attenuating acute AR.


Assuntos
Bortezomib/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Imunossupressores/uso terapêutico , Fatores Reguladores de Interferon/imunologia , Transplante de Rim , MicroRNAs/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Aloenxertos , Animais , Bortezomib/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/sangue , Feminino , Rejeição de Enxerto/imunologia , Imunossupressores/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/imunologia
15.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1544-1555, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326539

RESUMO

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Biotinilação , Bortezomib/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia
16.
Mol Med Rep ; 20(3): 2410-2418, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322176

RESUMO

The aim of the present study was to investigate the effect of bortezomib on heat shock protein 27 (HSP27) in multiple myeloma (MM) and provide a potential new target for clinical treatment. Peripheral blood was collected from 50 normal subjects and 50 patients with newly diagnosed MM and the expression of HSP27 was detected by ELISA. The changes of HSP27 after conventional vincristine, doxorubicin and dexamethasone (VAD) chemotherapy, and bortezomib plus VAD were compared. The effect of bortezomib on U266 cell proliferation and apoptosis was detected using a Cell Counting Kit­8 assay and Annexin V­FITC/propidium iodide double staining with flow cytometry. The content of HSP27 following bortezomib treatment was determined by ELISA. Western blot analysis and reverse transcription­quantitative PCR were used to detect the mRNA and protein expression of HSP27, Bax and Bcl­2. HSP27 expression was increased in patients with MM compared with healthy control subjects, and the expression was increased as the cancer progressed (P<0.05). Compared with the VAD chemotherapy group, the bortezomib plus VAD chemotherapy regimen significantly inhibited the expression of HSP27 (P<0.05), and the content of HSP27 was decreased in patients in which treatment was effective compared to those patients that exhibited disease progression (P<0.05). The efficacy of the treatment regimes was not associated with age or gender. Compared with the control group, bortezomib or OGX­427 (HSP27 inhibitor) treatment inhibited U266 cell proliferation, promoted U266 cell apoptosis (P<0.05) and significantly decreased HSP27 expression (P<0.05). Furthermore, the expression of HSP27 and Bcl­2 was significantly decreased, while the expression of Bax was increased by bortezomib and OGX­427 (P<0.05). There was no significant difference between the bortezomib and OGX­427 group in the in vitro analysis. HSP27 was positively correlated with Bcl­2 expression and negatively correlated with Bax expression in U266 cells. In conclusion, bortezomib promotes the apoptosis of MM cells, potentially by downregulating the expression of HSP27, providing a potential novel target for the clinical treatment of multiple myeloma.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib/uso terapêutico , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico/análise , Humanos , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/análise , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia
17.
ACS Appl Mater Interfaces ; 11(27): 23948-23956, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31192575

RESUMO

In this work, we prepared a novel cancer chemotherapeutic nanocarrier through the self-assembly of a mussel-derived, cancer-targeting peptide with a pH-sensitive conjugation of antitumor drugs. The biomimetic peptide was designed with a fluorescent molecule fluorescein isothiocyanate for imaging, a RGD sequence for cancer-targeting and tetravalent catechol groups for dynamic conjugation of the antitumor drug bortezomib via pH-cleavable boronic acid-catechol esters. Our study demonstrated that the peptide-based prodrug nanocarrier dramatically the enhanced specific cellular uptake and cytotoxicity toward human breast cancer cells in vitro in comparison with free drug and nontargeting control nanoparticles. Likewise, the prodrug nanocarrier showed improved therapeutic efficacy and low systematic toxicity in vivo. Considering highly biomimetic nature of the peptide-based nanocarriers, rapid drug release from the dynamically conjugated prodrugs, and convenience of introducing cancer-targeting activity onto this nanosystem, we believe our work would provide new ideas for the development of intelligent and biocompatible drug delivery systems to improve the chemotherapy efficacy in clinic. Furthermore, the pH-sensitive drug conjugation mechanism on peptide-based nanocarriers would provide a hint for the exploitation of dynamic prodrug strategies and the development of highly biocompatible nanocarriers using biogenic materials, e.g., the proteinogenic nanomaterials decorated with drugs through dynamic covalent chemistry.


Assuntos
Bivalves/química , Bortezomib , Portadores de Fármacos , Nanopartículas , Neoplasias Experimentais , Oligopeptídeos , Pró-Fármacos , Animais , Bortezomib/química , Bortezomib/farmacocinética , Bortezomib/farmacologia , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Neoplasia ; 21(7): 653-664, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31132676

RESUMO

The ubiquitin-proteasome system is elementary for cellular protein degradation and gained rising attention as a new target for cancer therapy due to promising clinical trials with bortezomib, the first-in class proteasome inhibitor meanwhile approved for multiple myeloma and mantle cell lymphoma. Both bortezomib and next-generation proteasome inhibitors mediate their effects by targeting the 20S core particle of the 26S proteasome. The novel small molecule inhibitor b-AP15 affects upstream elements of the ubiquitin-proteasome cascade by suppressing the deubiquitinase activity of both proteasomal regulatory 19S subunits and showed promising anticancer activity in preclinical models. Nonetheless, effects of inhibitors on the ubiquitin-proteasome system are not exclusively restricted to malignant cells: alteration of natural killer cell-mediated immune responses had already been described for drugs targeting either 19S or 20S proteasomal subunits. Moreover, it has been shown that bortezomib impairs dendritic cell (DC) phenotype and function at different levels. In the present study, we comparatively analyzed effects of bortezomib and b-AP15 on monocyte-derived DCs. In line with previous results, bortezomib exposure impaired maturation, antigen uptake, migration, cytokine secretion and immunostimulation, whereas treatment with b-AP15 had no compromising effects on these DC features. Our findings warrant the further investigation of b-AP15 as an alternative to clinically approved proteasome inhibitors in the therapy of malignancies, especially in the context of combinatorial treatment with DC-based immunotherapies.


Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Ubiquitina/genética , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Enzimas Desubiquitinantes/genética , Humanos , Monócitos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Piperidonas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia
19.
Mol Med Rep ; 19(6): 5023-5029, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059005

RESUMO

Multiple myeloma (MM) is an incurable disease; a better understanding of the molecular aspects of this hematological malignancy could contribute to the development of new treatment strategies and help to improve the survival rates of patients with MM. Previously, the methylation status of the deleted in colorectal cancer (DCC) gene was correlated with the survival rate of patients with MM, thus the main goal of this study was to understand DCC contribution to MM tumorigenesis, and to assess the impact of DCC inhibition in the MM response to treatment with bortezomib. Our results demonstrated that hypermethylation of the DCC promoter inhibits gene expression, and DCC silencing is significantly correlated with a reduction in cell viability and an increase in cell death induced by bortezomib. In conclusion, our results suggested that hypermethylation is an important mechanism of DCC expression regulation in MM and that the absence of DCC contributes to the enhanced sensitivity to treatment with bortezomib.


Assuntos
Bortezomib/farmacologia , Receptor DCC/metabolismo , Regulação para Baixo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptor DCC/antagonistas & inibidores , Receptor DCC/genética , Metilação de DNA/efeitos dos fármacos , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo
20.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088925

RESUMO

Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action.IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib's inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.


Assuntos
Antivirais/farmacologia , Bortezomib/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Internalização do Vírus/efeitos dos fármacos , Aciclovir/farmacologia , Animais , Núcleo Celular/metabolismo , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Prepúcio do Pênis/citologia , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Humanos , Masculino , Nucleocapsídeo/metabolismo , Células Vero
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