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Fibrous particles interact with cells and organisms in complex ways that can lead to cellular dysfunction, cell death, inflammation, and disease. The development of conductive transparent networks (CTNs) composed of metallic silver nanowires (AgNWs) for flexible touchscreen displays raises new possibilities for the intimate contact between novel fibers and human skin. Here, we report that a material property, nanowire-bending stiffness that is a function of diameter, controls the cytotoxicity of AgNWs to nonimmune cells from humans, mice, and fish without deterioration of critical CTN performance parameters: electrical conductivity and optical transparency. Both 30- and 90-nm-diameter AgNWs are readily internalized by cells, but thinner NWs are mechanically crumpled by the forces imposed during or after endocytosis, while thicker nanowires puncture the enclosing membrane and release silver ions and lysosomal contents to the cytoplasm, thereby initiating oxidative stress. This finding extends the fiber pathology paradigm and will enable the manufacture of safer products incorporating AgNWs.
Assuntos
Endossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Lisossomos/metabolismo , Nanofios/toxicidade , Animais , Linhagem Celular , Células Cultivadas , Condutividade Elétrica , Fibroblastos/metabolismo , Peixes , Humanos , Camundongos , Nanofios/química , Estresse Oxidativo , Prata/químicaRESUMO
The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R-like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted. SIGNIFICANCE STATEMENT: Multidrug resistance is one major challenge to cancer treatment. This study provides evidence that cancer cells overexpress 78-kDa glucose-regulated protein (GRP78) as a mechanism to acquire resistance to standard cancer therapies. A chromene-based small molecule, CXL146, selectively eliminates cancer cells with GRP78 overexpression via activating unfolded protein response-mediated apoptosis. Further characterization indicates that CXL146 and standard therapies complementarily target different populations of cancer cells, supporting the potential of CXL146 to overcome multidrug resistance in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Chaperona BiP do Retículo Endoplasmático , Células HL-60 , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Mitoxantrona/farmacologiaRESUMO
Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.
Assuntos
Células Eritroides/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas do Tecido Nervoso , Transporte Biológico Ativo , Sistemas CRISPR-Cas , Células Eritroides/citologia , Testes Genéticos , Células HEK293 , Heme/genética , Humanos , Células K562 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
PURPOSE: Understanding how differentiation, microenvironment, and hormonal milieu influence human breast cell susceptibility to malignant transformation will require the use of physiologically relevant in vitro systems. We sought to develop a 3D culture model that enables the propagation of normal estrogen receptor alpha (ER) + cells. METHODS: We tested soluble factors and protocols for the ability to maintain progenitor and ER + cells in cultures established from primary cells. Optimized conditions were then used to profile estrogen-induced gene expression changes in cultures from three pathology-free individuals. RESULTS: Long-term representation of ER + cells was optimal in medium that included three different TGFß/activin receptor-like kinase inhibitors. We found that omitting the BMP signaling antagonist, Noggin, enhanced the responsiveness of the PGR gene to estradiol exposure without altering the proportions of ER + cells in the cultures. Profiling of estradiol-exposed cultures showed that while all the cultures showed immediate and robust induction of PGR, LRP2, and IGFB4, other responses varied qualitatively and quantitatively across specimens. CONCLUSIONS: We successfully identified conditions for the maintenance and propagation of functional ER + cells from normal human breast tissues. We propose that these 3D cultures will overcome limitations of conventional 2D cultures of partially or fully transformed cell lines by sustaining normal endocrine function and growth regulation of the cell populations that comprise intact breasts.
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Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/metabolismo , Receptores de Estrogênio/genética , Esferoides Celulares , Ativação Transcricional , Células Tumorais CultivadasRESUMO
A highly versatile nanoplatform that couples mesoporous silica nanoparticles (MSNs) with an aerosol technology to achieve direct nanoscale delivery to the respiratory tract is described. This novel method can deposit MSN nanoparticles throughout the entire respiratory tract, including nasal, tracheobronchial and pulmonary regions using a water-based aerosol. This delivery method was successfully tested in mice by inhalation. The MSN nanoparticles used have the potential for carrying and delivering therapeutic agents to highly specific target sites of the respiratory tract. The approach provides a critical foundation for developing therapeutic treatment protocols for a wide range of diseases where aerosol delivery to the respiratory system would be desirable. FROM THE CLINICAL EDITOR: Delivery of drugs via the respiratory tract is an attractive route of administration. In this article, the authors described the design of mesoporous silica nanoparticles which could act as carriers for drugs. The underlying efficacy was successfully tested in a mouse model. This drug-carrier inhalation nanotechnology should potentially be useful in human clinical setting in the future.
Assuntos
Aerossóis , Nanopartículas , Dióxido de Silício/administração & dosagem , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar , Pneumopatias/terapia , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Dióxido de Silício/uso terapêuticoRESUMO
Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.
Assuntos
Sistemas CRISPR-Cas , Leucemia Mieloide Aguda , Humanos , Criança , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linhagem Celular , DNA Helicases/genéticaRESUMO
Metal-based drugs, such as cisplatin and auranofin, are used for the treatment of cancer and rheumatoid arthritis, respectively. Auranofin and other gold-derived compounds have been shown to possess anticancer, anti-inflammatory, antimicrobial, and antiparasitic activity in preclinical and clinical trials. Unlike platinum agents which are known to target DNA, the target of gold is not well elucidated. To better understand the targets and effects of gold agents in mammalian cells, we used a targeted CRISPR (ToxCRISPR) screen in K562 cancer cells to identify genes that modulate cellular sensitivity to gold. We synthesized a novel chiral gold(I) compound, JHK-21, with potent anticancer activity. Among the most sensitizing hits were proteins involved in mitochondrial carriers, mitochondrial metabolism, and oxidative phosphorylation. Further analysis revealed that JHK-21 induced inner mitochondria membrane dysfunction and modulated ATP-binding cassette subfamily member C (ABCC1) function in a manner distinct from auranofin. Characterizing the therapeutic effects and toxicities of metallodrugs in mammalian cells is of growing interest to guide future drug discovery, and cellular and preclinical/clinical studies.
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BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/genética , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Exorribonucleases/metabolismo , Edição de Genes/métodos , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/genética , Tratamento Farmacológico da COVID-19RESUMO
Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.
Assuntos
Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Piperazinas/uso terapêutico , Proteína bcl-X/metabolismo , Animais , Antineoplásicos/farmacologia , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos NOD , Neoplasias Pancreáticas/patologia , Piperazinas/farmacologia , GencitabinaRESUMO
Organochlorine pesticides (OCPs) are persistent pollutants linked to diverse adverse health outcomes. Environmental exposure to OCPs has been suggested to negatively impact the immune system but their effects on cellular antiviral responses remain unknown. Transcriptomic analysis of N27 rat dopaminergic neuronal cells unexpectedly detected high level expression of genes in the interferon (IFN)-related antiviral response pathways including the IFN-induced protein with tetratricopeptide repeats 1 and 2 (Ifit1/2) and the MX Dynamin Like GTPases Mx1 and Mx2. Interestingly, treatment of N27 cells with dieldrin markedly downregulated the expression of many of these genes. Dieldrin exterted a similar effect in inhibiting IFIT2 and MX1 gene expression in human SH-SY5Y neuronal cells induced by an RNA viral mimic, polyinosinic: polycytidylic acid (poly I:C) and IFIT2/3 gene expression in human pulmonary epithelial cells exposed to human influenza H1N1 virus. Mechanistically, dieldrin induced a rapid rise in levels of intracellular reactive oxygen species (iROS) and a decrease in intracellular glutathione (GSH) levels in SH-SY5Y cells. Treatment with N-acetylcysteine, an antioxidant and GSH biosynthesis precursor, effectively blocked both dieldrin-induced increases in iROS and its inhibition of poly I:C-induced upregulation of IFIT and MX gene expression, suggesting a role for intracellular oxidative status in dieldrin's modulation of antiviral gene expression. This study demonstrates that dieldrin modulates key genes of the cellular innate immune responses that are normally involved in the host's cellular defense against viral infections. Our findings have potential relevance to understanding the organismal effects of environmentally persistent organochlorine contaminants on the mammalian cellular immune system.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Praguicidas , Animais , Antivirais , Dieldrin/toxicidade , Neurônios Dopaminérgicos , Expressão Gênica , Humanos , Interferons , Praguicidas/toxicidade , RatosRESUMO
Formaldehyde (FA), a ubiquitous environmental pollutant, is classified as a Group I human carcinogen by the International Agency for Research on Cancer. Previously, we reported that FA induced hematotoxicity and chromosomal aneuploidy in exposed workers and toxicity in bone marrow and hematopoietic stem cells of experimental animals. Using functional toxicogenomic profiling in yeast, we identified genes and cellular processes modulating eukaryotic FA cytotoxicity. Although we validated some of these findings in yeast, many specific genes, pathways and mechanisms of action of FA in human cells are not known. In the current study, we applied genome-wide, loss-of-function CRISPR screening to identify modulators of FA toxicity in the human hematopoietic K562 cell line. We assessed the cellular genetic determinants of susceptibility and resistance to FA at 40, 100 and 150 µM (IC10, IC20 and IC60, respectively) at two time points, day 8 and day 20. We identified multiple candidate genes that increase sensitivity (e.g. ADH5, ESD and FANC family) or resistance (e.g. FASN and KDM6A) to FA when disrupted. Pathway analysis revealed a major role for the FA metabolism and Fanconi anemia pathway in FA tolerance, consistent with findings from previous studies. Additional network analyses revealed potential new roles for one-carbon metabolism, fatty acid synthesis and mTOR signaling in modulating FA toxicity. Validation of these novel findings will further enhance our understanding of FA toxicity in human cells. Our findings support the utility of CRISPR-based functional genomics screening of environmental chemicals.
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Anemia de Fanconi , Hipersensibilidade Respiratória , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Anemia de Fanconi/genética , Formaldeído/efeitos adversos , Formaldeído/toxicidade , HumanosRESUMO
Organochlorine pesticides, once widely used, are extremely persistent and bio-accumulative in the environment. Epidemiological studies have implicated that environmental exposure to organochlorine pesticides including dieldrin is a risk factor for the development of Parkinson's disease. However, the pertinent mechanisms of action remain poorly understood. In this study, we carried out a genome-wide (Brunello library, 19 114 genes, 76 411 sgRNAs) CRISPR/Cas9 screen in human dopaminergic SH-SY5Y neuronal cells exposed to a chronic treatment (30 days) with dieldrin to identify cellular pathways that are functionally related to the chronic cellular toxicity. Our results indicate that dieldrin toxicity was enhanced by gene disruption of specific components of the ubiquitin proteasome system as well as, surprisingly, the protein degradation pathways previously implicated in inherited forms of Parkinson's disease, centered on Parkin. In addition, disruption of regulatory components of the mTOR pathway which integrates cellular responses to both intra- and extracellular signals and is a central regulator for cell metabolism, growth, proliferation, and survival, led to increased sensitivity to dieldrin-induced cellular toxicity. This study is one of the first to apply a genome-wide CRISPR/Cas9-based functional gene disruption screening approach in an adherent neuronal cell line to globally decipher cellular mechanisms that contribute to environmental toxicant-induced neurotoxicity and provides novel insight into the dopaminergic neurotoxicity associated with chronic exposure to dieldrin.
Assuntos
Sistemas CRISPR-Cas , Dieldrin , Neurônios Dopaminérgicos/efeitos dos fármacos , Praguicidas , Linhagem Celular , Dieldrin/toxicidade , Humanos , Praguicidas/toxicidadeRESUMO
Central predictions of climate warming models include increased climate variability and increased severity of heat waves. Physiological acclimatization in populations across large-scale ecological gradients in habitat temperature fluctuation is an important factor to consider in detecting responses to climate change related increases in thermal fluctuation. We measured in vivo cardiac thermal maxima and used microarrays to profile transcriptome heat and cold stress responses in cardiac tissue of intertidal zone porcelain crabs across biogeographic and seasonal gradients in habitat temperature fluctuation. We observed acclimatization dependent induction of heat shock proteins, as well as unknown genes with heat shock protein-like expression profiles. Thermal acclimatization had the largest effect on heat stress responses of extensin-like, beta tubulin, and unknown genes. For these genes, crabs acclimatized to thermally variable sites had higher constitutive expression than specimens from low variability sites, but heat stress dramatically induced expression in specimens from low variability sites and repressed expression in specimens from highly variable sites. Our application of ecological transcriptomics has yielded new biomarkers that may represent sensitive indicators of acclimatization to habitat temperature fluctuation. Our study also has identified novel genes whose further description may yield novel understanding of cellular responses to thermal acclimatization or thermal stress.
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Aclimatação/fisiologia , Anomuros/genética , Perfilação da Expressão Gênica , Miocárdio/metabolismo , Animais , Anomuros/fisiologia , Clima , Análise de Sequência com Séries de Oligonucleotídeos , Estações do Ano , TemperaturaRESUMO
Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.
Assuntos
Acetaldeído/toxicidade , Sistemas CRISPR-Cas , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Neoplasias/induzido quimicamente , Neoplasias/genética , Proteínas/genética , Proteínas Supressoras de Tumor/genética , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Adutos de DNA/genética , Adutos de DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Células K562 , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas/metabolismo , Medição de Risco , Proteínas Supressoras de Tumor/metabolismoRESUMO
Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (AsIII) is also an effective chemotherapeutic agent. The current use of AsIII in chemotherapy is limited to acute promyelocytic leukemia (APL). However, AsIII was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPR-based approach to identify modulators of AsIII toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased AsIII tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to AsIII whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to AsIII. Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to AsIII. Our data suggest a model in which an intracellular interaction between selenium and AsIII may impact intracellular AsIII levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of AsIII in CML cells. Targeting such processes simultaneously with AsIII treatment could potentiate AsIII in CML therapy.
Assuntos
Antineoplásicos/farmacologia , Trióxido de Arsênio/farmacologia , Perfilação da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Sistemas CRISPR-Cas , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edição de Genes , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Transdução de Sinais , Selenito de Sódio/farmacologia , Fatores de Tempo , TranscriptomaRESUMO
BACKGROUND: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. OBJECTIVES: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). METHODS: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. RESULTS: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. CONCLUSION: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. CITATION: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM, Yaswen P, Vulpe CD, Leitman DC. 2016. Parabens and human epidermal growth factor receptor ligand cross-talk in breast cancer cells. Environ Health Perspect 124:563-569; http://dx.doi.org/10.1289/ehp.1409200.
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Estrogênios/toxicidade , Parabenos/toxicidade , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/metabolismo , Genes myc , Humanos , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Nanowires (NWs), high-aspect-ratio nanomaterials, are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from nanoparticles. We carried out a comprehensive evaluation of the physicochemical stability of four silver nanowires (AgNWs) of two sizes and coatings and their toxicity to Daphnia magna . Inorganic aluminum-doped silica coatings were less effective than organic poly(vinyl pyrrolidone) coatings at preventing silver oxidation or Ag(+) release and underwent a significant morphological transformation within 1 h following addition to low ionic strength Daphnia growth media. All AgNWs were highly toxic to D. magna but less toxic than ionic silver. Toxicity varied as a function of AgNW dimension, coating, and solution chemistry. Ag(+) release in the media could not account for observed AgNW toxicity. Single-particle inductively coupled plasma mass spectrometry distinguished and quantified dissolved and nanoparticulate silver in microliter-scale volumes of Daphnia magna hemolymph with a limit of detection of approximately 10 ppb. The silver levels within the hemolymph of Daphnia exposed to both Ag(+) and AgNW met or exceeded the initial concentration in the growth medium, indicating effective accumulation during filter feeding. Silver-rich particles were the predominant form of silver in hemolymph following exposure to both AgNWs and Ag(+). Scanning electron microscopy imaging of dried hemolymph found both AgNWs and silver precipitates that were not present in the AgNW stock or the growth medium. Both organic and inorganic coatings on the AgNW were transformed during ingestion or absorption. Pathway, gene ontology, and clustering analyses of gene expression response indicated effects of AgNWs distinct from ionic silver on Daphnia magna .
Assuntos
Daphnia/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Nanofios/toxicidade , Prata/toxicidade , Alumínio/química , Animais , Perfilação da Expressão Gênica , Hemolinfa/efeitos dos fármacos , Dose Letal Mediana , Oxigênio/química , Povidona/química , Dióxido de Silício/química , Prata/química , Compostos de Prata/química , Compostos de Prata/toxicidade , Testes de Toxicidade , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidadeRESUMO
BACKGROUND: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. METHODOLOGY/PRINCIPAL FINDINGS: A set of approximately 30K unique sequences (UniSeqs) representing approximately 19K clusters were generated from approximately 98K high quality ESTs from a set of tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66% of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases. CONCLUSIONS/SIGNIFICANCE: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics.
Assuntos
Decápodes/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Animais , Análise por Conglomerados , Biologia Computacional/métodos , DNA Complementar/química , DNA Complementar/genética , Bases de Dados Genéticas , Biblioteca Gênica , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNARESUMO
Selenium (Se) phytovolatilization, the process by which plants metabolize various inorganic or organic species of Se (e.g. selenate, selenite, and Se-methionine [Met]) into gaseous Se forms (e.g. dimethylselenide), is a potentially important means of removing Se from contaminated environments. Before attempting to genetically enhance the efficiency of Se phytovolatilization, it is essential to elucidate the enzymatic pathway involved and to identify its rate-limiting steps. The present research tested the hypothesis that S-adenosyl-L-Met:L-Met S-methyltransferase (MMT) is the enzyme responsible for the methylation of Se-Met to Se-methyl Se-Met (SeMM). To this end, we identified and characterized an Arabidopsis T-DNA mutant knockout for MMT. The lack of MMT in the Arabidopsis T-DNA mutant plant resulted in an almost complete loss in its capacity for Se volatilization. Using chemical complementation with SeMM, the presumed enzymatic product of MMT, we restored the capacity of the MMT mutant to produce volatile Se. Overexpressing MMT from Arabidopsis in Escherichia coli, which is not known to have MMT activity, produced up to 10 times more volatile Se than the untransformed strain when both were supplied with Se-Met. Thus, our results provide in vivo evidence that MMT is the key enzyme catalyzing the methylation of Se-Met to SeMM.