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Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.
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COVID-19 , Ensaios de Triagem em Larga Escala , Pandemias , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Antivirais/uso terapêutico , Antivirais/farmacologiaRESUMO
Acute kidney injury (AKI) is a major public health concern with significant morbidity and mortality and no current treatments beyond supportive care and dialysis. Preclinical studies have suggested that heme-oxygenase-1 (HO-1), an enzyme that catalyzes the breakdown of heme, has promise as a potential therapeutic target for AKI. Clinical trials involving HO-1 products (biliverdin, carbon monoxide, and iron), however, have not progressed beyond the Phase ½ level. We identified small-molecule inducers of HO-1 that enable us to exploit the full therapeutic potential of HO-1, the combination of its products, and yet-undefined effects of the enzyme system. Through cell-based, high-throughput screens for induction of HO-1 driven by the human HO-1 promoter/enhancer, we identified two novel small molecules and broxaldine (an FDA-approved drug) for further consideration as candidate compounds exhibiting an Emax ≥70% of 5 µM hemin and EC50 <10 µM. RNA sequencing identified shared binding motifs to NRF2, a transcription factor known to regulate antioxidant genes, including HMOX1. In vitro, the cytoprotective function of the candidates was assessed against cisplatin-induced cytotoxicity and apoptosis. In vivo, delivery of a candidate compound induced HO-1 expression in the kidneys of mice. This study serves as the basis for further development of small-molecule HO-1 inducers as preventative or therapeutic interventions for a variety of pathologies, including AKI.
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Arsenicals belong to the class of chemical warfare agents known as vesicants, which are highly reactive, toxic and cause robust inflammatory response. Cutaneous exposure to arsenicals causes a wide range of systemic organ damage, beginning with cutaneous injuries, and later manifest multi-organ damage and death. Thus, the development of suitable antidotes that can effectively block injury following exposure to these agents is of great importance. Bromodomain 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) family, plays crucial role in regulating transcription of inflammatory, proliferation and cell cycle genes. In this context, the development of potent small molecule inhibitors of BRD4 could serve as potential antidotes for arsenicals. Herein, we describe the synthesis and biological evaluation of a series of compounds.
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Arsenicais , Anti-Inflamatórios/química , Antídotos/farmacologia , Arsenicais/farmacologia , Arsenicais/uso terapêutico , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Liquid biopsy is becoming an innovative tool in precision oncology owing to its noninvasive identification of biomarkers circulating in the body fluid at various time points for continuous and real-time analysis of disease progression. MicroRNAs in blood exosomes are identified as a new promising class of potential biomarkers for cancer diagnostics and prognostics. Conventional detection of blood exosomal microRNAs need multiple-step, complicated, costly, and time-consuming sample preparation of exosomes isolation and RNA extract, which affect the accuracy and reproducibility of analytical results. In this work, we set up an in situ quantitative analysis of human plasma exosomal miR-1246 by a probe of 2'-O-methyl and phosphorothioate modified molecular beacon. The probe has outstanding nuclease resistance in highly active RNase A/T1/I, which makes it stable for direct application in blood samples. With rapid rupture of exosomes membrane by Triton X-100, the probe can enter exosomes to specifically target miR-1246 exhibiting quantitative fluorescent signals. Using the output signals as a diagnostic marker, we differentiated 33 breast cancer patients from 37 healthy controls with 97.30% sensitivity and 93.94% specificity at the best cutoff. The blood biopsy is simple without extracting plasma exosomes and their nucleic acids content, time-saving in about 2 h of total analysis process, and microvolumes needed for plasma sample, suggesting its good potential to clinical application.
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Neoplasias da Mama , Exossomos , MicroRNAs , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biópsia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Exossomos/química , Exossomos/genética , Humanos , Biópsia Líquida , MicroRNAs/genética , Medicina de Precisão , Reprodutibilidade dos TestesRESUMO
Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.
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Proliferação de Células/genética , Melanoma/genética , Fatores de Crescimento Neural/genética , Receptor ErbB-4/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Mutação com Ganho de Função/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Fosforilação/genética , Receptor ErbB-4/agonistas , Receptor ErbB-4/antagonistas & inibidores , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
BACKGROUND: This study evaluated whether obese male mice exposed to di-(2-ethylhexyl) phthalate (DEHP) showed synergistic effects on testosterone levels and the potential underlying mechanism. METHODS: Forty-eight male mice were assigned to six groups for 12-week treatments as follows: normal, DEHP100, diet-induced obesity (DIO), DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300. Serum hormone levels, including testosterone (T), luteinizing hormone (LH), and leptin, were detected by ELISA. The levels of Ob-R, kisspeptin, and GPR54 protein expression in hypothalamus and testicular tissues were measured by western blot. RESULTS: There were significantly lower levels of serum T and LH, higher levels of serum leptin and Ob-R, and kisspeptin and GPR54 protein expression were reduced in hypothalamus and testicular tissues in the DIO and DEHP groups compared with controls. Moreover, serum T and leptin levels were more severe in the combined DIO and DEHP exposure group than in the single exposure groups. Serum LH levels and GPR54 expression in the testis were significantly decreased in DIO + DEHP300 mice compared with DIO mice (p < 0.05). CONCLUSION: Obesity- and DEHP-only exposure had adverse effects on testosterone levels in mice, which may be due to high leptin levels and decreased Ob-R, kisspeptin, and GPR54 expression. Obesity combined with DEHP exposure had an additive adverse effect on testosterone levels in mice. One of the potential mechanisms is higher leptin levels and decreased GPR54 expression in the testes.
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Dietilexilftalato/toxicidade , Hipotálamo/metabolismo , Kisspeptinas/análise , Obesidade/metabolismo , Receptores de Kisspeptina-1/análise , Testículo/metabolismo , Testosterona/sangue , Animais , Leptina/sangue , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The Foxo3 gene, belonging to the forkhead family, is one of the classes of transcription factors characterized by a forkhead DNA-binding domain, which usually considered being a cancer suppressor gene. Circ-Foxo3 is a circular structure which connects the 3'end to the 5'end. Scholars detected that circ-Foxo3 could compete with Foxo3 for binding to some miRNAs. METHODS: In this study, we will test the expression of Foxo3 and circ-Foxo3 in de novo acute myeloid leukemia (AML) patients to explore the relationship between Foxo3 gene and circ-Foxo3. All the de novo AML samples and normal control samples was measured by real-time quantitative PCR. A receiver operating characteristic curve was conducted to differentiate AML patients from control people. Association of Foxo3 expression and overall survival was conducted by Kaplan-Meier survival analysis. RESULTS: We found that the expression of Foxo3 gene in de novo patients was significantly lower than control samples (P = 0.009). Meanwhile, circ-Foxo3 also expressed lower in de novo AML patients than in control samples (P = 0.040). In different classifications, this trend could be observed more remarkably. In non-M3 patients, the Foxo3 high patients' survival time was longer than Foxo3 low patients (P = 0.002). Besides, in non-favorable risk groups, patients with low expression of Foxo3 had longer survival time than Foxo3 high patients (P = 0.004). Furthermore, in normal Karyotypic patients, the overall survival time of patients with high-expressed Foxo3 was significantly longer than those with low expression (P = 0.034). Besides, Pearson analysis was also conducted between these two genes in AML patients. Results revealed that they were positively correlated (R = 0.63, P < 0.001). CONCLUSION: In conclusion, we found that low expression of circ-Foxo3 and Foxo3 were frequent in AML patients, and patients with high expression of Foxo3 often had a trend of better prognosis.
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Proteína Forkhead Box O3/metabolismo , Leucemia Mieloide Aguda/metabolismo , RNA Circular/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Criança , Feminino , Proteína Forkhead Box O3/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.
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Antineoplásicos , Benzotiazóis , Naftiridinas , Neoplasias , RNA Neoplásico/biossíntese , RNA Ribossômico/biossíntese , Ribossomos/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzotiazóis/química , Benzotiazóis/farmacologia , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Naftiridinas/química , Naftiridinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
We herein report an efficient Ag+ and S2- dual sensing scenario by a three-dimensional (3D) Cu-based metal-organic framework [Cu(Cdcbp)(bpea)] n (MOF 1, H3CdcbpBr = 3-carboxyl-(3,5-dicarboxybenzyl)-pyridinium bromide, bpea = 1,2-di(4-pyridinyl)ethane) shielded with a 5-carboxytetramethylrhodamine (TAMRA)-labeled C-rich single-stranded DNA (ss-probe DNA, P-DNA) as a fluorescent probe. The formed MOF-DNA probe, denoted as P-DNA@1, is able to sequentially detect Ag+ and S2- in one pot, with detection limits of 3.8 nM (for Ag+) and 5.5 nM (for S2-), which are much more lower than the allowable Ag+ (0.5 µM) and S2- (0.6 µM) concentration in drinking water as regulated by World Health Organization (WHO). The detection method has been successfully applied to sense Ag+ and S2- in domestic, lake, and mineral water with satisfactory recoveries ranging from 98.2 to 107.3%. The detection mechanism was further confirmed by molecular simulation studies.
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Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 µL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption.
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Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Biomarcadores Tumorais/sangue , Biópsia , Linhagem Celular Tumoral , Exossomos/metabolismo , Feminino , Humanos , Cinética , Células MCF-7 , Mamografia/métodos , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , TermodinâmicaRESUMO
Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl4-treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl4-induced liver damage. YGL also suppressed the increase of TGF-ß1, TNF-α, IL-1ß, IL-6, IL-4 and IL-17â¯A induced by CCl4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl4-induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-ß1/Smad signaling pathway and modulating the balance among IL-4, IL-17â¯A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis.
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Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Matriz Extracelular/efeitos dos fármacos , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citoproteção , Relação Dose-Resposta a Droga , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Masculino , Fosforilação , Proteólise , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
A 2D metal-organic framework (MOF) of {[Cu(Dcbb)(Bpe)]·Cl} n (1, H2DcbbBr = 1-(3,5-dicarboxybenzyl)-4,4'-bipyridinium bromide, Bpe = trans-1,2-bis(4-pyridyl)ethylene)) has been prepared. MOF 1 associates with the thymine-rich (T-rich), single-stranded probe DNA (ss-DNA, denoted as P-DNA) labeled with fluorophore FAM (FAM = carboxyfluorescein) and quenches the FAM emission to give a nonemissive P-DNA@1 hybrid (off state). The P-DNA in the hybrid subsequently captures the Hg2+ to give a rigid double-stranded DNA featuring T-Hg2+-T motif (ds-DNA@Hg2+) and detach from MOF 1, triggering the recovery of the FAM fluorescence (on state). Upon subsequent addition of I-, Hg2+ was further sequestrated from the ds-DNA@Hg2+ duplex, driven by the stronger Hg-I coordination. The released P-DNA is resorbed by MOF 1 to regain the initial P-DNA@1 hybrid (off state). The P-DNA@1 sensor thus detects Hg2+ and I- sequentially via a fluorescence "off-on-off" mechanism. The sensor is highly selective and sensitive, yielding detection limits of 3.2 and 3.3 nM, respectively. The detection process was conformed by circular dichroism (CD) and the detection mechanism was verified by fluorescence anisotropy, binding constant, and simulation of the binding free energy at each stage.
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Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , Iodetos/análise , Mercúrio/análise , Estruturas Metalorgânicas/química , Cobre/química , DNA de Cadeia Simples/genética , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Ligantes , Limite de Detecção , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência , Timina/químicaRESUMO
FUS1 is a tumor suppressor gene that has been found to be frequently lost in a variety of solid tumors. In this study, we aimed to investigate the expression status of the FUS1 gene in acute myeloid leukemia (AML), as well as its clinical significance. We further explored the correlation between the expression of FUS1 and miR-378 in AML. We detected expression of the FUS1 transcript in bone marrow mononuclear cells from 23 controls and 158 newly diagnosed AML patients by real-time quantitative polymerase chain reaction. Downregulated FUS1 expression was found in 139 out of 158 (87.97%) AML cases; this rate was significantly lower than that in all 23 controls (p = 0.012). Receiver operating characteristic curve analysis revealed that the FUS1 transcript level could discriminate AML patients from controls effectively (area under the ROC curve = 0.663). Kaplan-Meier analysis demonstrated that non-M3-AML patients with a low FUS1 expression had a shorter overall survival (p = 0.049) and leukemia-free survival (p = 0.051) than those with a high FUS1 expression. Furthermore, we studied the correlation between the expression of FUS1 and miR-378 in 53 newly diagnosed AML patients. We found that the correlation coefficient was -0.346, which showed that FUS1 and miR-378 were negatively correlated in AML patients (p = 0.011). These results indicate that the low expression of FUS1 is a common molecular event in AML.
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Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Estudos de Casos e Controles , Criança , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Prognóstico , Curva ROC , Adulto JovemRESUMO
Chemerin is dysregulation in numerous solid cancers. However, only little is known about the role of chemerin in acute myeloid leukemia (AML). In this study, we aimed to investigate the expression and clinical significance of recently described chemerin in acute myeloid leukemia (AML). The expression of chemerin in 149 patients with de novo AML and 35 normal controls was quantified by Real-time quantitative PCR (RQ-PCR). Chemerin was down-expressed in AML compared with controls (P=0.042). A receiver operating characteristic (ROC) curve revealed that chemerin expression could differentiate patients with AML from control subjects (AUC=0.611, 95% CI: 0.490-0.732; P=0.042) respectively. The cohort of AML patients was divided into two groups according to the cut-off value of 0.0826 (79% sensitivity and 54% specificity, respectively). In addition, the AML patients with low chemerin expression had significantly shorter overall survival (OS) than those with high chemerin expression (P=0.049). Moreover, multivariate survival analysis confirmed that chemerin was an independent prognostic factor for AML patients. In conclusion, downregulation of chemerin might be a useful diagnostic and prognostic factor for AML patients.
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OBJECTIVES: This study was intended to investigate the expression status of Vimentin 2p (VIM 2p), a pseudogene of Vimentin, and further analyze its clinical significance in AML patients. METHODS: Real-time quantitative PCR (RQ-PCR) was employed to explore the expression status of VIM 2p in 128 patients with de novo AML and 36 healthy controls. RESULTS: The expression level of VIM 2p was significantly decreased compared with healthy controls (P< 0.001). The patients with low VIM 2p expression were identified in 93 of 128 (73%) of AML patients. No significant differences could be observed in sex, age, blood parameters, FAB/WHO subtypes, karyotype risks and ten gene mutations (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3 A, C/EBPA, N/K-RAS and U2AF1) between VIM 2p low-expressed and high-expressed patients (P> 0.05). Patients with low VIM 2p expression had significantly shorter overall survival (OS) than those with high VIM 2p expression in whole AML cases (median 7 vs. 13 months, respectively, P= 0.032), besides cytogenetically normal AML (CN-AML) and non-M3 AML cohort (P= 0.042 and 0.045, respectively). CONCLUSIONS: These findings indicate that VIM 2p down-regulation is a common event in AML and may be associated with poor clinical outcome.
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Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Pseudogenes , Vimentina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biomarcadores Tumorais , Estudos de Casos e Controles , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Cariótipo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Prognóstico , Curva ROC , Análise de Sobrevida , Adulto JovemRESUMO
The BMI1P1 levels of 144 de novo AML patients and 36 healthy donors were detected by real-time quantitative PCR (RQ-PCR). BMI1P1 was significantly down-regulated in AML compared with control (P < 0.001). A receiver operating characteristic (ROC) curve revealed that BMI1P1 expression could differentiate patients with AML from control subjects (AUC = 0.895, 95% CI: 0.835-0.954, P < 0.001). The percentage of blasts in bone marrow (BM) was significantly lower in BMI1P1 high-expressed group versus low-expressed group (P = 0.008). BMI1P1 high-expressed cases had significantly higher complete remission (CR) than BMI1P1 low-expressed cases (P = 0.023). Furthermore, Kaplan-Meier demonstrated that both whole AML cohort and non-M3-AML patients with low BMI1P1 expression showed shorter leukemia free survival (LFS, P = 0.002 and P = 0.01, respectively) and overall survival (OS, P < 0.001 and P = 0.011, respectively) than those with high BMI1P1 expression. Multivariate analysis also showed that BMI1P1 over-expression was an independent favorable prognostic factor for OS in both whole and non-M3 cohort of AML patients (HR = 0.462, 95% CI = 0.243-0.879, P = 0.019 and HR = 0.483, 95% CI = 0.254-0.919, P = 0.027). To further investigate the significance of BMI1P1 expression in the follow-up of AML patients, we monitored the BMI1P1 level in 26 de novo AML patients and found that the BMI1P1 level increased significantly from the initial diagnosis to post-CR (P < 0.001). These results indicated that BMI1P1 might contribute to the diagnosis of AML and the assessment of therapeutic effect.
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Leucemia Mieloide Aguda/genética , Complexo Repressor Polycomb 1/genética , Pseudogenes , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
Mitotic kinesin Eg5 is an attractive anticancer drug target. Discovery of Eg5 inhibitors has been focused on targeting the 'monastrol-binding site'. However, acquired drug resistance has been reported for such inhibitors. Therefore, identifying new Eg5 inhibitors which function through a different mechanism(s) could complement current drug candidates and improve drug efficacy. In this study, we explored a novel allosteric site of Eg5 and identified new Eg5 inhibitors through structure-based virtual screening. Experiments with the saturation-transfer difference NMR demonstrated that the identified Eg5 inhibitor SRI35566 binds directly to Eg5 without involving microtubules. Moreover, SRI35566 and its two analogs significantly induced monopolar spindle formation in colorectal cancer HCT116 cells and suppressed cancer cell viability and colony formation. Together, our findings reveal a new allosteric regulation mechanism of Eg5 and a novel drug targeting site for cancer therapy.
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Cinesinas/antagonistas & inibidores , Regulação Alostérica , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Microscopia Confocal , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 µM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment.
Assuntos
Descoberta de Drogas , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Tiadiazóis/química , Tiadiazóis/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Humanos , Cinesinas/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Tiadiazóis/farmacologiaRESUMO
BACKGROUND: Dysregulation of miR-675 has been found in a variety of solid tumors. MiR-675 has been suggested as having both oncogenic and tumor suppression properties in cancer. However, there is no evidence whether miR-675 is involved in breast cancer. The objective of this study was to evaluate the expression status of miR-675 and its clinical relevance in breast cancer patients. METHODS: The expression level of miR-675 was detected in 100 breast cancer patients and 38 cancer-free controls using real-time quantitative PCR. The clinicopathological characteristics of miR-675 in breast cancer were also investigated. All statistical analyses were performed using SPSS 20.0. RESULTS: The study showed that miR-675 was significantly up-regulated in breast cancer patients compared with controls (P < 0.01). There was no significant difference in age, lymph nodes stage, ER status and PR status between patients with and without miR-675 over-expression (P > 0.05). The frequency of miR-675 over-expression was higher in the patients of histological grade I-II than in others (50% versus 9%, P = 0.011). The expression level of miR-675 had a high correlation with miR-24/93/98/378 in breast cancer patients. CONCLUSIONS: Taken together, our study demonstrated that miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues might serve as a good source for biomarker discovery and breast cancer validation.
RESUMO
Kinesin-like protein KIFC1, a normally nonessential kinesin motor, plays a critical role in centrosome clustering in cancer cells and is essential for the survival of cancer cells. Herein, we reported that KIFC1 expression is up-regulated in breast cancer, particularly in estrogen receptor negative, progesterone receptor negative and triple negative breast cancer, and is not associated with epidermal growth factor receptor 2 status. In addition, KIFC1 is highly expressed in all 8 tested human breast cancer cell lines, but is absent in normal human mammary epithelial cells and weakly expressed in 2 human lung fibroblast lines. Moreover, KIFC1 silencing significantly reduced breast cancer cell viability. Finally, we found that PJ34, a potent small molecule inhibitor of poly(ADP-ribose) polymerase, suppressed KIFC1 expression and induced multipolar spindle formation in breast cancer cells, and inhibited cell viability and colony formation within the same concentration range, suggesting that KIFC1 suppression by PJ34 contributes to its anti-breast cancer activity. Together, these results suggest that KIFC1 is a novel promising therapeutic target for breast cancer.