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1.
Artigo em Inglês | MEDLINE | ID: mdl-32942346

RESUMO

Introduction: Bipolar disorder is a complex mood disorder characterized by a chronic and subtle course of fluctuating manic/hypomanic and depressive symptoms. Cariprazine, a dopamine D3-preferring D3/D2 receptor partial agonist with serotonin 5-HT1A receptor partial agonist and serotonin 5-HT2A antagonist properties, is approved to treat manic and depressive episodes of bipolar disorder. Post hoc analyses evaluated efficacy across symptoms in bipolar depression. Methods: Pooled data were analyzed from 3 phase 2 or 3, randomized, double-blind, placebo-controlled studies of adults with bipolar disorder and a major depressive episode. Mean change from baseline to week 6 in Montgomery-Asberg Depression Rating Scale (MADRS) total score and individual item scores were analyzed in individual dose groups (1.5 mg/d, 3 mg/d) and overall cariprazine (1.5-3 mg/d). Pooled safety was evaluated via adverse events. Results: A significantly greater difference in mean change from baseline in MADRS total score was seen for each cariprazine dose group versus placebo (least squares mean difference vs placebo: 1.5-3 mg/d = -2.6, 1.5 mg/d = -2.8, 3 mg/d = -2.4) (P < .001 all). Significant differences versus placebo were seen on all individual MADRS items except inner tension for the overall cariprazine group (P < .05). Cariprazine was generally well tolerated. Conclusions: Cariprazine demonstrated broad efficacy across symptoms of depression in bipolar disorder. In previous post hoc analyses, cariprazine also demonstrated broad efficacy across manic symptoms, suggesting that it is effective across the wide range of symptoms on the bipolar spectrum. A 1.5-mg/d starting dose and slow titration resulted in lower rates of some adverse events in the bipolar depression studies versus the mania studies. Trial Registration: ClinicalTrials.gov identifiers: NCT01396447, NCT02670538, NCT02670551.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Depressivo Maior/tratamento farmacológico , Neurotransmissores/farmacologia , Piperazinas/farmacologia , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurotransmissores/administração & dosagem , Neurotransmissores/efeitos adversos , Piperazinas/administração & dosagem , Piperazinas/efeitos adversos , Resultado do Tratamento
2.
Nat Commun ; 11(1): 4051, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792548

RESUMO

The RNA genome of the human immunodeficiency virus (HIV) is reverse-transcribed into DNA and integrated into the host genome, resulting in latent infections that are difficult to clear. Here we show an approach to eradicate HIV infections by selective elimination of host cells harboring replication-competent HIV (SECH), which includes viral reactivation, induction of cell death, inhibition of autophagy and the blocking of new infections. Viral reactivation triggers cell death specifically in HIV-1-infected T cells, which is promoted by agents that induce apoptosis and inhibit autophagy. SECH treatments can clear HIV-1 in >50% mice reconstituted with a human immune system, as demonstrated by the lack of viral rebound after withdrawal of treatments, and by adoptive transfer of treated lymphocytes into uninfected humanized mice. Moreover, SECH clears HIV-1 in blood samples from HIV-1-infected patients. Our results suggest a strategy to eradicate HIV infections by selectively eliminating host cells capable of producing HIV.


Assuntos
Infecções por HIV/prevenção & controle , HIV-1/patogenicidade , Animais , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Azepinas/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Humanos , Camundongos , Organofosfatos/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Pirimidinonas/farmacologia , RNA Viral/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Triazóis/farmacologia
3.
Nat Commun ; 11(1): 4249, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843618

RESUMO

Aberrant cell cycle machinery and loss of the CDKN2A tumor suppressor locus make CDK4/6 a potential target in pancreatic ductal adenocarcinoma (PDAC). However, a vast majority of PDAC cases do not harbor a durable response to monotherapy of CDK4/6 inhibitor. Utilizing remote loading to co-encapsulate CDK4/6 inhibitor palbociclib (PAL) and an autophagy inhibitor hydroxychloroquine (HCQ), we demonstrate a ratiometrically designed mesoporous silica nanoformulation with synergistic efficacy in subcutaneous and orthotopic PDAC mouse models. The synergism is attributed to the effective intratumoral buildup of PAL/HCQ, which otherwise exhibit distinctly different circulatory and biodistribution profile. PAL/HCQ co-delivery nanoparticles lead to the most effective shrinkage of PDAC compared to various controls, including free drug mixture. Immunohistochemistry reveals that PAL/HCQ co-delivery nanoparticles trigger anti-apoptotic pathway after repetitive intravenous administrations in mice. When combined with a Bcl inhibitor, the performance of co-delivery nanoparticles is further improved, leading to a long-lasting anti-PDAC effect in vivo.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/química , Hidroxicloroquina/farmacologia , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , Piperazinas/administração & dosagem , Piperazinas/química , Piperazinas/farmacologia , Piridinas/administração & dosagem , Piridinas/química , Piridinas/farmacologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Resultado do Tratamento
4.
PLoS One ; 15(8): e0237196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764777

RESUMO

Current antidepressant therapy has several disadvantages related to the properties of antidepressants. Considering their unfavourable features, the process of searching for new antidepressant drugs with better safety and tolerability requires consistent efforts and many complementary studies. Serotonin 5-HT1A receptor is considered as an interesting target of antidepressant therapy. In the present study, the intrinsic activity at different signaling pathways coupled to serotonin 5-HT1A receptor, antidepressant-like and pharmacokinetic properties, and the safety profile of two novel imidazopurine-2,4-dione derivatives, namely compounds AZ-853 (8-(4-(4-(2-fluorophenyl)piperazin-1-yl)butyl)-1,3-dimethyl-1H- imidazo[2,1-f]purine-2,4(3H,8H)-dione) and AZ-861 (1,3-dimethyl-8-(4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)butyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione), were studied in animal models through in vitro and in vivo experiments. We demonstrated that AZ-853 and AZ-861, which structurally differ by one substituent and its placement in the phenyl ring, showed varied functional, pharmacological, and pharmacokinetic properties as well as side effect profiles. AZ-861 exhibited stronger agonistic action in all functional assays. After acute and repeated administration in mice, both compounds showed antidepressant-like activity in the forced swim test, which was partially mediated by 5-HT1A receptor activation. AZ-853 showed a more potent antidepressant-like effect, presumably due to its better penetration into brain structures. Both compounds did not show anticholinergic properties, but after repeated administration, they induced weak sedation and lipid metabolism disturbances without affecting serum glucose level. The stronger α1-adrenolytic effect of AZ-853 is responsible for decreased systolic blood pressure, and in contrast to AZ-861, AZ-853 induced weight gain in mice. The interesting comparative pharmacological profiles of AZ-853 and AZ-861 encourage to conduct further experiments to fully understand their mechanisms and differences in action.


Assuntos
Antidepressivos/farmacologia , Depressão/tratamento farmacológico , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Animais , Antidepressivos/química , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Depressão/diagnóstico , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Piperazinas/química , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Agonistas do Receptor 5-HT1 de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/uso terapêutico , Relação Estrutura-Atividade , Ganho de Peso/efeitos dos fármacos
5.
Anticancer Res ; 40(7): 3781-3792, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32620617

RESUMO

BACKGROUND/AIM: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. MATERIALS AND METHODS: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. RESULTS: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. CONCLUSION: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.


Assuntos
Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cães , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Syk/metabolismo
6.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 1018-1022, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701246

RESUMO

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 µg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). CONCLUSIONS: Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.


Assuntos
Edema Encefálico , Lesões Encefálicas Traumáticas , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz , Animais , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indazóis/farmacologia , Indazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
7.
Life Sci ; 257: 118086, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679147

RESUMO

AIMS: To investigate the role of PP2A in calcified aortic valve disease (CAVD). MATERIALS AND METHODS: The expressions of PP2A subunits were detected by real-time polymerase chain reaction (RT-PCR) and western blot in aortic valves from patients with CAVD and normal controls, the activities of PP2A were analyzed by commercial assay kit at the same time. Aortic valve calcification of mice was evaluated through histological and echocardiographic analysis. ApoE-/- mice and ApoE-/- mice injected intraperitoneally with PP2A inhibitor LB100 were fed a high-cholesterol diet for 24 weeks. Immunofluorescent staining was used to locate the cell-type in which PP2A activity was decreased, the PP2A activity of valvular interstitial cells (VICs) treated with osteogenic induction medium was assessed by western blot and commercial assay kit. After changing the activity of VICs through pharmacologic and genetic intervention, the osteoblast differentiation and mineralization were assessed by western blot and Alizarin Red staining. Finally, the mechanism was clarified by using several specific inhibitors. KEY FINDINGS: PP2A activity was decreased both in calcified aortic valves and human VICs under osteogenic induction. The PP2A inhibitor LB100 aggravated the aortic valve calcification of mice. Furthermore, PPP2CA overexpression inhibited osteogenic differentiation of VICs, whereas PPP2CA knockdown promoted the process. Further study revealed that the ERK/p38 MAPKs signaling pathways mediated the osteogenic differentiation of VICs induced by PP2A inactivation. SIGNIFICANCE: This study demonstrated that PP2A plays an important role in CAVD pathophysiology, PP2A activation may provide a novel strategy for the pharmacological treatment of CAVD.


Assuntos
Estenose da Valva Aórtica/fisiopatologia , Valva Aórtica/patologia , Apolipoproteínas E/genética , Calcinose/fisiopatologia , Osteogênese/genética , Proteína Fosfatase 2/genética , Animais , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/genética , Calcinose/genética , Diferenciação Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piperazinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Parasitol Res ; 119(9): 3093-3097, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32591863

RESUMO

The efficacy of pyrantel pamoate, pyrvinium pamoate, ivermectin, and piperazine citrate against pinworm in cockroach was evaluated. Laboratory-reared German cockroaches naturally infected with Blatticola blattae were treated with the anthelmintics and necropsied at 3 to 35 days after treatment. Ivermectin at over 5 ppm and piperazine citrate at over 2000 ppm killed all the treated cockroaches. Pinworms were still detected in cockroaches given lower concentration of the aforementioned drugs. Administration of pyrantel pamoate (100-1000 ppm) and pyrvinium pamoate (2000 ppm) did not kill the cockroaches, and no pinworms were detected at 3 and 17 days after treatment. Thus, pyrantel pamoate and pyrvinium pamoate were found to be effective for deworming B. blattae in the German cockroaches, without causing mortality for the host. Our results showed that anthelmintics selection is essential for eradication of pinworms in cockroaches because of the toxicity for the host such as ivermectin or piperazine citrate. This is the first report of piperazine citrate toxicity in cockroaches.


Assuntos
Anti-Helmínticos/farmacologia , Baratas/parasitologia , Enterobíase/parasitologia , Enterobius/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Enterobíase/tratamento farmacológico , Enterobius/fisiologia , Humanos , Ivermectina/farmacologia , Piperazinas/farmacologia , Pamoato de Pirantel/farmacologia
9.
Anticancer Res ; 40(5): 2509-2514, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366395

RESUMO

BACKGROUND/AIM: Matrix-producing breast carcinoma (MPBC) is a rare and usually aggressive triple-negative breast cancer (TNBC). In this study, we determined drug sensitivity for a triple-negative MPBC, without BRCA mutations, in a patient-derived orthotopic xenograft (PDOX) model. MATERIALS AND METHODS: The MPBC PDOX model was established in the left 2nd mammary gland of nude mouse by implantation of the patient tumor using surgical orthotopic implantation (SOI). We randomized MPBC PDOX mice into 5 groups (n=5 mice/per treatment group) when the tumor volume reached 80 mm3: G1, control-no treatment; G2, bevacizumab [intra-peritoneal (i.p.), weekly, for 2 weeks]; G3, vinorelbine (i.p., weekly, for 2 weeks); G4, olaparib (oral., daily, for 2 weeks); G5, eribulin [intravenous (i.v.), weekly, for 2 weeks]. The mice in each treatment group were sacrificed on day 15. Tumor volume and body weight were measured once/week. RESULTS: The MPBC PDOX model was resistant to olaparib (p=0.22). The MPBC PDOX model treated with bevacizumab and vinorelbine showed significantly suppressed tumor growth compared to the untreated group (p=0.005 and 0.002, respectively). However, only eribulin regressed the tumor (p=0.0001). Eribulin was more effective than olaparib (p=0.0001), bevacizumab (p=0.0025) and vinorelbine (p=0.0061). CONCLUSION: Eribulin has clinical potential as treatment for triple-negative MPBC patients that are resistant to a PARP inhibitor such as olaparib.


Assuntos
Bevacizumab/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Furanos/farmacologia , Cetonas/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Vinorelbina/farmacologia , Adulto , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Life Sci ; 255: 117846, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32470451

RESUMO

AIMS: Compared to normal cells, tumor cells maintain higher concentrations of reactive oxygen species (ROS) to support proliferation, invasion, and metastasis. Chemotherapeutic drugs often induce tumor cell apoptosis by increasing intracellular ROS concentrations to highly toxic levels. ABT737, which inhibits the apoptosis regulator B cell lymphoma 2 (Bcl2), increases the sensitivity of ovarian cancer cells to chemotherapeutic drugs by regulating the glucose metabolism, but the underlying mechanisms remain unclear. Therefore, we aimed to determine whether ABT737 promoted H2O2-induced tumor cell apoptosis by reversing glycolysis in ovarian cancer cells. MAIN METHODS: SKOV3 ovarian cancer cells were treated with H2O2, ABT737, or both. Cell viability was compared using methyl thiazolyl tetrazolium (MTT), and flow cytometry was used to detect differences in apoptosis, ROS, and mitochondrial membrane potential. The relative expression levels of proteins associated with apoptosis and the glucose metabolism were measured using immunoblotting. Finally, glucose uptake and lactate secretion were measured using kits and compared. KEY FINDINGS: ABT737 downregulated proteins associated with glucose uptake (GLUT1) and glycolysis (LHDA, PKM2 and HK2) via the Sirt3-HIF1α axis, reducing glucose uptake and lactate secretion in SKOV3 cells. This reversed glycolysis in the tumor cells, and promoted H2O2-induced apoptosis. SIGNIFICANCE: The Bcl2 inhibitor ABT737 enhanced the anti-tumor effect of oxidative stress by reversing the Warburg effect in ovarian cancer cells, providing powerful theoretical support for further clinical applications of Bcl2 inhibitors.


Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Nitrofenóis/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/metabolismo
11.
Nat Commun ; 11(1): 2573, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444794

RESUMO

Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, here we develop a method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly (ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer classification system is 95% and 95% (95% confidence intervals: 77-100% and 82-99%), respectively. We classify the functional impact of 186 BRCA2 VUSs with our computational pipeline, resulting in the classification of 126 variants as normal/likely normal, 23 as intermediate, and 37 as abnormal/likely abnormal. We further describe a simplified, on-demand annotation system that could be used as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/genética , Ensaios de Triagem em Larga Escala/métodos , Teorema de Bayes , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional/métodos , Éxons , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Mutação de Sentido Incorreto , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução Genética
12.
Hum Cell ; 33(3): 801-809, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32388810

RESUMO

Endometrial cancer (EC) is one of the most common cancers among females worldwide. Advanced stage patients of EC have poor prognosis. Inevitable side effects and treatment tolerance of chemotherapy for EC remain to be addressed. Our results in this study showed that EC cells with higher tumor necrosis factor receptor-associated factor 4 (TRAF4) expression have lower sensitivity to poly ADP-ribose polymerase 1 (PARP1) inhibitors. Upon TRAF4 knockdown, the colony numbers of EC cells were markedly down-regulated, and the markers of DNA double-strand breakage were significantly up-regulated after the treatment of olaparib, a PARP1 inhibitor. TRAF4 knockdown reduced the phosphorylation of protein kinase B (Akt), promoted DNA double-strand breakage, and decreased levels of DNA repair related proteins, including phosphorylated-DNA-dependent protein kinase (p-DNA-PK) and RAD51 recombinase (RAD51). In addition, TRAF4's effect on the sensitivity of EC cells to olaparib was further found to be mainly mediated by Akt phosphorylation. Moreover, in vivo results showed that TRAF4 knockdown enhanced the sensitivity of EC to PARP1 inhibitors using a mouse xenograft model. Collectively, our data suggest that combined application of TRAF4 knockdown and PARP1 inhibition can be used as a promising strategy for synthetic lethality in EC treatment.


Assuntos
Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Técnicas de Silenciamento de Genes , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Neoplasias do Endométrio/patologia , Feminino , Expressão Gênica , Humanos , Camundongos , Terapia de Alvo Molecular , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator 4 Associado a Receptor de TNF/genética , Fator 4 Associado a Receptor de TNF/metabolismo , Células Tumorais Cultivadas
13.
Prostate ; 80(11): 831-849, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32449814

RESUMO

INTRODUCTION: Prostate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin-linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real-time polymerase chain reaction (RT-PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide), the integrin α2ß1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide), or the integrin α4ß1/α9ß1 inhibitor BOP (N-(benzenesulfonyl)- l-prolyl- l-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt). RESULTS: Western blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, ß1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT-PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate-specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and ß1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency-dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT-3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT-3033 at 32 Hz). The thromboxane A2 analog U46619-induced concentration-dependent contractions, which were inhibited by Cpd22 and BTT-3033 (around 67% by Cpd22 and 39% by BTT-3033 at 30 µM U46619). Endothelin-1 induced concentration-dependent contractions, which were not affected by Cpd22 or BTT-3033. Noradrenaline and the α1 -adrenergic agonists methoxamine and phenylephrine-induced concentration-dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT-3033. BOP did not change EFS- or agonist-induced contraction. CONCLUSIONS: Integrin α2ß1 and ILK inhibitors inhibit neurogenic and thromboxane A2 -induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.


Assuntos
Integrina alfa2beta1/antagonistas & inibidores , Músculo Liso/efeitos dos fármacos , Próstata/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Dipeptídeos/farmacologia , Humanos , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Piperazinas/farmacologia , Próstata/metabolismo , Próstata/fisiologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Tromboxano A2/metabolismo , Vasoconstritores/farmacologia
14.
Nat Commun ; 11(1): 2350, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393766

RESUMO

BET inhibitors are promising therapeutic agents for the treatment of triple-negative breast cancer (TNBC), but the rapid emergence of resistance necessitates investigation of combination therapies and their effects on tumor evolution. Here, we show that palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule inhibitor, synergize with the BET inhibitor JQ1 in TNBC lines. High-complexity DNA barcoding and mathematical modeling indicate a high rate of de novo acquired resistance to these drugs relative to pre-existing resistance. We demonstrate that the combination of JQ1 and palbociclib induces cell division errors, which can increase the chance of developing aneuploidy. Characterizing acquired resistance to combination treatment at a single cell level shows heterogeneous mechanisms including activation of G1-S and senescence pathways. Our results establish a rationale for further investigation of combined BET and CDK4/6 inhibition in TNBC and suggest novel mechanisms of action for these drugs and new vulnerabilities in cells after emergence of resistance.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Proteínas/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Azepinas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Clonais , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Modelos Biológicos , Mutação/genética , Paclitaxel/farmacologia , Piperazinas/farmacologia , Ploidias , Proteínas/metabolismo , Piridinas/farmacologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Resultado do Tratamento , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
15.
Science ; 368(6486)2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241924

RESUMO

The success of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerase-1/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Benzimidazóis/química , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Humanos , Isoindóis/química , Isoindóis/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Domínios Proteicos
16.
Nat Commun ; 11(1): 1669, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245950

RESUMO

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.


Assuntos
Antígeno B7-H1/genética , Linfócitos do Interstício Tumoral/imunologia , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/genética , Adulto , Idoso , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Prognóstico , Regiões Promotoras Genéticas/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Análise Serial de Tecidos , Ativação Transcricional/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidade , Regulação para Cima/efeitos dos fármacos
17.
Nucleic Acids Res ; 48(9): 4928-4939, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297953

RESUMO

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Antineoplásicos/farmacologia , Dano ao DNA , Proteínas de Ligação a DNA/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Galinhas , Cromatina/enzimologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Mutagênicos/toxicidade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(17): 9497-9507, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32300005

RESUMO

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Disponibilidade Biológica , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Óxido Nítrico , Óxido Nítrico Sintase Tipo III/genética , Piperazinas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Ligação Proteica , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , para-Aminobenzoatos/farmacologia
19.
J Cancer Res Clin Oncol ; 146(7): 1659-1670, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32279088

RESUMO

PURPOSE: Desmoplastic small round cell tumors (DSRCTs) are highly malignant and very rare soft tissue sarcomas with a high unmet need for new therapeutic options. Therefore, we examined poly(ADP-ribose) polymerase 1 (PARP1) and Schlafen-11 (SLFN11) expression in DSRCT tumor tissue and the combination of PARP inhibitor olaparib with the alkylating agent temozolomide (TMZ) in a preclinical DSRCT model. METHODS: PARP1 and SLFN11 have been described as predictive biomarkers for response to PARP inhibition. Expression of PARP1 and SLFN11 was assessed in 16 and 12 DSRCT tumor tissue samples, respectively. Effects of single-agent olaparib, and olaparib and TMZ combination treatment were examined using the preclinical JN-DSRCT-1 model. In vitro, single-agent and combination treatment effects on cell viability, the cell cycle, DNA damage and apoptosis were examined. Olaparib and TMZ combination treatment was also assessed in vivo. RESULTS: PARP1 and SLFN11 expression was observed in 100% and 92% of DSRCT tumor tissues, respectively. Olaparib treatment reduced cell viability and cell migration in a dose-dependent manner in vitro. Drug synergy between olaparib and TMZ was observed in vitro and in vivo. Combination treatment led to a cell-cycle arrest and induction of DNA damage and apoptosis, even when combined at low dosages. CONCLUSION: We show high PARP1 and SLFN11 expression in DSRCT tumor material and antitumor effects following olaparib and TMZ combination treatment in a preclinical DSRCT model. This suggests that olaparib and TMZ combination treatment could be a potential treatment option for DSRCTs.


Assuntos
Antineoplásicos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Temozolomida/farmacologia , Adolescente , Adulto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Tumor Desmoplásico de Pequenas Células Redondas/tratamento farmacológico , Tumor Desmoplásico de Pequenas Células Redondas/etiologia , Tumor Desmoplásico de Pequenas Células Redondas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
20.
Nat Commun ; 11(1): 1145, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123179

RESUMO

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.


Assuntos
Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Anticorpos de Domínio Único/química , Sítio Alostérico , Sítios de Ligação , Técnicas Biossensoriais , Cristalografia por Raios X , AMP Cíclico/metabolismo , Dinorfinas/química , Dinorfinas/farmacologia , Células HEK293 , Humanos , Medições Luminescentes/métodos , Piperazinas/química , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Conformação Proteica , Pirrolidinas/química , Pirrolidinas/farmacologia , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/metabolismo , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia
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