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The present investigation reports embryo-induced modifications in the epithelial cells of the endometrium in a primate species. In vivo, epithelial cell response to the embryonic signals was assessed at the embryo attachment stage in the gestational uterus of bonnet monkeys (Macaca radiata) and in vitro response was investigated by treating human endometrial epithelial cell line (Ishikawa) with human embryo conditioned media (CM). Endometrial epithelial (EE) cells at the embryo attachment stage in bonnet monkeys revealed higher proliferation accompanied by significant up regulation (p < 0.05) in the expression of estrogen receptor (ER)α and down regulation (p < 0.05) in ERß expression. Further gestational EE cells showed higher (p < 0.001) expression of mucin-1, except in the embryo attachment site. Also, observed were significantly higher expression (p < 0.05) and altered cytoplasmic distribution of α(v) and ß(3) integrins, when compared to non-pregnant animals. In pregnant animals, the embryo attachment zone showed differential expression of immunoreactive integrins as compared to the non-attachment zone. This suggested the role of embryo secreted factors in modulation of the epithelial cell profile. In vitro studies partially supported this assumption. Significantly higher proliferation (p < 0.05), as well as increased expression of ERα, integrin ß(3) and mucin-1 (p < 0.05) were observed in Ishikawa cells, on stimulation with CM. Taken together, these results indicated the proliferation and modulation in the expression of estrogen receptors and cell adhesion molecules in the EE cells; at the embryo attachment stage in bonnet monkeys. Further it is likely that embryo secreted factors contribute to some of these modifications in EE cells. This report is the first account of discrete cellular events, which occur in the uterine epithelium, at the embryo attachment stage in a primate species.
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Embrião de Mamíferos/metabolismo , Endométrio/metabolismo , Células Epiteliais/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Endométrio/embriologia , Feminino , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Macaca radiata , Mucina-1/metabolismoRESUMO
Graft verus host disease (GVHD) following Liver transplantation is rare life threatening complication with very high mortality rate around 85%. Due to increased recognition of this condition management approach is rapidly evolving due to newer diagnostic methods and drugs. Etiology, risk factors, pathogenesis, preventive strategies, management approach and newer drugs are discussed. We present our experience of 2 cases from a large cohort of 1052 Liver transplant operations over a decade.
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Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.
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Neuroblastoma , Neurofibromina 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Recidiva Local de Neoplasia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.
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Neoplasias de Cabeça e Pescoço , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Epirregulina/metabolismo , Inibidores Enzimáticos/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.
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The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGA-MPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 microM and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 microM for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos , Etoposídeo/administração & dosagem , Nanopartículas , Antineoplásicos Fitogênicos/metabolismo , Linhagem Celular Tumoral , Etoposídeo/metabolismo , Humanos , Microscopia ConfocalRESUMO
The present investigation was aimed at developing PEGylated PLGA nanoparticles of cytarabine. PLGA Nanoparticles were prepared by modified nanoprecipitation method, optimized for mean particle size (152 ± 6 nm) and entrapment efficiency (41.1 ± 0.8%) by a 3² factorial design. The PEGylated PLGA nanoparticles of cytarabine had a zeta potential of -7.5 ± 1.3 mV and sustained the release of cytarabine for 48 h by Fickian diffusion. The IC50 values for L1210 cells were 6.5, 5.3, and 2.2 µM for cytarabine, cytarabine loaded PLGA nanoparticles and cytarabine loaded PLGA-mPEG nanoparticles respectively. Confocal microscopy and flow cytometry showed that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Biodistribution studies showed that the PEGylated nanoparticles of cytarabine were present in significantly higher concentrations in blood circulation as well as in brain and bones and avoided RES uptake as compared to the free drug.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Citarabina/administração & dosagem , Preparações de Ação Retardada/química , Ácido Láctico/química , Nanopartículas/química , Polietilenoglicóis/química , Ácido Poliglicólico/química , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Citarabina/efeitos adversos , Citarabina/sangue , Citarabina/farmacocinética , Feminino , Humanos , Leucemia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-DawleyRESUMO
Bedside barcode technology is used during medication administration to ensure patient safety. This study evaluated the workflow variables related to a bedside barcode technology-based medication administration process. A time-and-motion technique was used to assess the observational episodes related to medication administration conducted by registered nurses. In an observational episode, nurses spent adequate time in "documenting medications" and "giving medications." Nurses were primarily engaged in tasks at the patient's bedside.
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Processamento Eletrônico de Dados , Sistemas de Medicação no Hospital , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Fluxo de Trabalho , Estudos Transversais , Humanos , Pesquisa em Avaliação de Enfermagem , Registros de Enfermagem , Estudos Prospectivos , Qualidade da Assistência à Saúde , Fatores de Tempo , Estudos de Tempo e MovimentoRESUMO
Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Óxidos N-Cíclicos/farmacologia , Indolizinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Compostos de Piridínio/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Óxidos N-Cíclicos/administração & dosagem , Sinergismo Farmacológico , Feminino , Amplificação de Genes , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Indolizinas/administração & dosagem , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Oxazóis/administração & dosagem , Oxazóis/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Compostos de Piridínio/administração & dosagem , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Distribuição Aleatória , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.
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The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.
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Benzazepinas/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Histonas/antagonistas & inibidores , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Fenilenodiaminas/farmacologia , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Pirimidinas/farmacologia , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Transcriptoma , Animais , Apoptose , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2-dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Animais , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proliferação de Células , Ciclofosfamida/administração & dosagem , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Sulfonamidas/administração & dosagem , Topotecan/administração & dosagem , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. SIGNIFICANCE: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.
Assuntos
Ferro/farmacologia , Neuroblastoma/tratamento farmacológico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Auranofina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Amplificação de Genes , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa/metabolismo , Humanos , Ferro/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oxazóis/farmacologia , Oxazóis/uso terapêutico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Sulfassalazina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensitivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disappointing, suggesting an underappreciated resistance mechanism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug combination capable of rescuing PARP inhibitor activity. EXPERIMENTAL DESIGN: We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models. RESULTS: We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/XL inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone by venetoclax is insufficient to sensitize Ewing sarcoma cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL in Ewing sarcoma survival. CONCLUSIONS: These data reveal BCL-2 and BCL-XL act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sarcoma de Ewing/genética , Proteína bcl-X/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/metabolismoRESUMO
Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658-72. ©2018 AACR.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Terapia Combinada , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Camundongos , Modelos Biológicos , Terapia de Alvo Molecular , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Telomerase activation is one of the key mechanisms that allow cells to bypass replicative senescence. Telomerase activity is primarily regulated at the level of transcription of its catalytic unit- hTERT. Prostate cancer (PCa), akin to other cancers, is characterized by high telomerase activity. Existing data suggest that hTERT expression and telomerase activity are positively regulated by androgenic stimuli in androgen-dependent prostate cancer (ADPC) cells. A part of the present study reaffirmed this by demonstrating a decline in the hTERT expression and telomerase activity on "loss of AR" in ADPC cells. The study further addressed 2 unresolved queries, i) whether AR-mediated signaling is of any relevance to hTERT expression in castration-resistant prostate cancer (CRPC) and ii) whether this signaling involves EGR1. Our data suggest that AR-mediated signaling negatively regulates hTERT expression in CRPC cells. Incidental support for the possibility of EGR1 being a regulator of hTERT expression in PCa was provided by i) immunolocalization of hTERT and EGR1 proteins in the same cell type (secretory epithelium) of PCa and BPH tissues; ii) significantly (p< 0.001) higher levels of both these proteins in CRPC (PC3 and DU145), compared with ADPC (LNCaP) cells. A direct evidence for the role of EGR1 in hTERT expression was evident by a significant (p<0.0001) decrease in the hTERT transcript levels in the EGR1-silenced CRPC cells. Further, "gain of AR" led to a significant reduction in the levels of hTERT and EGR1 in CRPC cells. However, restoration of EGR1 levels prevented the decline in the hTERT transcript levels in these cells. Taken together, our data indicate that AR regulates the expression of EGR1, which in turn acts as a positive regulator of hTERT expression in CRPC cells. Thus, AR exerts an inhibitory effect on hTERT expression and telomerase activity by modulating EGR1 levels in CRPC cells.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Telomerase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial-to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was initiated to explore whether ZEB2 expression differs in prostate cancer (PCa, n=7) and benign prostatic hyperplasia (BPH, n=7) tissues. In PCa tissues, the levels of both immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher (P<0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P<0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P<0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection of AR and ZEB2 cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen-dependent cells and as a negative regulator in AI PCa cells.
Assuntos
Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , MicroRNAs/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Imunofluorescência , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Hiperplasia Prostática/tratamento farmacológico , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/química , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Cicatrização , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
PURPOSE: The effect of bar-code-assisted medication administration (BCMA) on nurses' activities in an intensive care unit was evaluated. METHODS: A prospective, observational, time-motion study was conducted by considering two approaches to medication administration in an intensive care unit: paper-based medication administration (PBMA) and BCMA. The time spent on nursing activities was measured using a prevalidated time-motion observation instrument and categorized based on workflow factors such as direct patient care, indirect patient care, administration, and miscellaneous or other. A descriptive analysis was conducted with the amount of time spent on each of the nursing activities. A multivariate analysis of covariance was conducted to assess the difference between the two approaches for the amount of time spent on various categorized nursing activities. Covariates included in the analysis were patient characteristics, medication administration characteristics, and number of nurses involved in medication administration. RESULTS: A total of 101 PBMAs and 151 BCMAs were reviewed. The mean duration of total medication administration time was higher in the BCMA phase compared with the PBMA phase, as was the mean time spent on direct patient care activity. However, nurses spent less time on administration activity during BCMA. Statistical analysis revealed that the medication administration approach (BCMA versus PBMA) had a significant effect on time spent on direct patient care and medication administration activities. CONCLUSION: The implementation of BCMA led to a reduction in the time spent by nurses on medication administration activities and increased the time spent on direct patient care activities.