RESUMEN
Peripheral artery disease is a major atherosclerotic disease that is associated with poor outcomes such as limb loss, cardiovascular morbidity, and death. Artificial intelligence (AI) has seen increasing integration in medicine, and its various applications can optimize the care of peripheral artery disease (PAD) patients in diagnosis, predicting patient outcomes, and imaging interpretation. In this review, we introduce various AI applications such as natural language processing, supervised machine learning, and deep learning, and we analyze the current literature in which these algorithms have been applied to PAD.
Asunto(s)
Procesamiento de Lenguaje Natural , Enfermedad Arterial Periférica , Valor Predictivo de las Pruebas , Humanos , Enfermedad Arterial Periférica/terapia , Enfermedad Arterial Periférica/diagnóstico por imagen , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatología , Aprendizaje Profundo , Aprendizaje Automático Supervisado , Diagnóstico por Computador , Toma de Decisiones Clínicas , Pronóstico , Inteligencia Artificial , Técnicas de Apoyo para la DecisiónRESUMEN
BACKGROUND: Novel fusion transcripts (FTs) caused by chromosomal rearrangement are common factors in the development of cancers. In the current study, the authors used massively parallel RNA sequencing to identify new FTs in colon cancers. METHODS: RNA sequencing (RNA-Seq) and TopHat-Fusion were used to identify new FTs in colon cancers. The authors then investigated whether the novel FT nuclear receptor subfamily 5, group A, member 2 (NR5A2)-Kelch-like family member 29 FT (KLHL29FT) was transcribed from a genomic chromosomal rearrangement. Next, the expression of NR5A2-KLHL29FT was measured by quantitative real-time polymerase chain reaction in colon cancers and matched corresponding normal epithelia. RESULTS: The authors identified the FT NR5A2-KLHL29FT in normal and cancerous epithelia. While investigating this transcript, it was unexpectedly found that it was due to an uncharacterized polymorphic germline insertion of the NR5A2 sequence from chromosome 1 into the KLHL29 locus at chromosome 2, rather than a chromosomal rearrangement. This germline insertion, which occurred at a population frequency of 0.40, appeared to bear no relationship to cancer development. Moreover, expression of NR5A2-KLHL29FT was validated in RNA specimens from samples with insertions of NR5A2 at the KLHL29 gene locus, but not from samples without this insertion. It is interesting to note that NR5A2-KLH29FT expression levels were significantly lower in colon cancers than in matched normal colonic epithelia (P =.029), suggesting the potential participation of NR5A2-KLHL29FT in the origin or progression of this tumor type. CONCLUSIONS: NR5A2-KLHL29FT was generated from a polymorphism insertion of the NR5A2 sequence into the KLHL29 locus. NR5A2-KLHL29FT may influence the origin or progression of colon cancer. Moreover, researchers should be aware that similar FTs may occur due to transchromosomal insertions that are not correctly annotated in genome databases, especially with current assembly algorithms. Cancer 2017;123:1507-1515. © 2017 American Cancer Society.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Colon/metabolismo , Neoplasias del Colon/genética , Mutagénesis Insercional , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Neoplasias del Colon/metabolismo , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
Esophageal squamous cell carcinoma (ESCC) has a poor prognosis due to high lymphatic metastatic recurrence rates after Ivor Lewis esophagectomy. We sought to investigate the correlation between tumor necrosis factor alpha-induced protein 8 (TNFAIP8) expression and postoperative lymphatic recurrence in patients with pN0 ESCC. One hundred twenty-two patients with pN0 ESCC undergoing Ivor Lewis esophagectomy were enrolled in this study. TNFAIP8 overexpression was found in 73 (59.8 %) tumor specimens. The 3-year lymphatic metastatic recurrence rate among TNFAIP8-overexpressing patients was significantly higher than in TNFAIP8-negative patients (p = 0.003). Multivariate Cox regression identified TNFAIP8 overexpression as an independent risk factor for lymphatic recurrence (p = 0.048). TNFAIP8 messenger RNA (mRNA) levels were significantly higher in patients with lymphatic recurrence than in patients without tumor recurrence (p = 0.019). Stable silencing of TNFAIP8 expression in ESCC-derived cells (Eca109) reduced proliferation, motility, and invasion and induced apoptosis. In addition, transient silencing of TNFAIP8 expression decreased cell motility and invasion and increased apoptosis in a second ESCC-derived cell line (KYSE150). Taken together, these findings suggest that TNFAIP8 overexpression is a potential biomarker to identify pN0 ESCC patients at higher risk of lymphatic recurrence who may benefit from adjuvant therapy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Metástasis Linfática/patología , Recurrencia Local de Neoplasia/patología , Adulto , Anciano , Proteínas Reguladoras de la Apoptosis/análisis , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago , Esofagectomía , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Reacción en Cadena de la Polimerasa , Modelos de Riesgos Proporcionales , Factores de RiesgoRESUMEN
OBJECTIVES: Long non-coding RNAs (lncRNA) have been shown to play important roles in the development and progression of cancer. However, functional lncRNAs and their downstream mechanisms are largely unknown in the molecular pathogenesis of oesophageal adenocarcinoma (EAC) and its progression. DESIGN: lncRNAs that are abnormally upregulated in EACs were identified by RNA-sequencing analysis, followed by quantitative RT-PCR (qRTPCR) validation using tissues from 25 EAC patients. Cell biological assays in combination with small interfering RNA-mediated knockdown were performed in order to probe the functional relevance of these lncRNAs. RESULTS: We discovered that a lncRNA, HNF1A-AS1, is markedly upregulated in human primary EACs relative to their corresponding normal oesophageal tissues (mean fold change 10.6, p<0.01). We further discovered that HNF1A-AS1 knockdown significantly inhibited cell proliferation and anchorage-independent growth, suppressed S-phase entry, and inhibited cell migration and invasion in multiple in vitro EAC models (p<0.05). A gene ontological analysis revealed that HNF1A-AS1 knockdown preferentially affected genes that are linked to assembly of chromatin and the nucleosome, a mechanism essential to cell cycle progression. The well known cancer-related lncRNA, H19, was the gene most markedly inhibited by HNF1A-AS1 knockdown. Consistent to this finding, there was a significant positive correlation between HNF1A-AS1 and H19 expression in primary EACs (p<0.01). CONCLUSIONS: We have discovered abnormal upregulation of a lncRNA, HNF1A-AS1, in human EAC. Our findings suggest that dysregulation of HNF1A-AS1 participates in oesophageal tumorigenesis, and that this participation may be mediated, at least in part, by modulation of chromatin and nucleosome assembly as well as by H19 induction.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Expresión Génica , ARN Largo no Codificante/genética , ARN Largo no Codificante/fisiología , Adenocarcinoma/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Neoplasias Esofágicas/patología , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño , Puntos de Control de la Fase S del Ciclo Celular/genética , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: Alterations in methylation of protein-coding genes are associated with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Dysregulation of noncoding RNAs occurs during carcinogenesis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS: We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS: BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS: BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.
Asunto(s)
Adenocarcinoma/genética , Esófago de Barrett/genética , ADN de Neoplasias/genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Microfilamentos/genética , ARN Largo no Codificante/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Metilación de ADN , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , Proteínas de Microfilamentos/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genéticaRESUMEN
The incidence of gastroesophageal cancers is increasing each year, but despite much research, their molecular mechanisms are incompletely understood. microRNAs (miRNAs) are noncoding RNAs that have been associated with gastroesophageal carcinogenesis. We review the involvement of miRNAs in gastric and esophageal cancers and their mechanisms of regulation, effects on gene expression, and biological functions. Many miRNAs are dysregulated in gastroesophageal cancer cells via alterations in transcription, epigenetic features, or copy number of the genes that encode them. Each type of gastroesophageal tumor has a unique gene expression profile. miRNAs contribute to gastroesophageal carcinogenesis by altering expression of oncogenes and tumor suppressors to affect cell proliferation, apoptosis, and motility and invasion. A number of miRNAs, including circulating miRNAs, have been associated with tumor type or stage, or patient survival, and might be developed as diagnostic or prognostic markers. Greater understanding of the roles of miRNAs in gastroesophageal carcinogenesis could provide insights into the mechanisms of tumor development and identify therapeutic targets.
Asunto(s)
Neoplasias Esofágicas/genética , MicroARNs , Neoplasias Gástricas/genética , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/fisiopatología , Neoplasias Esofágicas/terapia , Perfilación de la Expresión Génica , Humanos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/fisiopatología , Neoplasias Gástricas/terapiaRESUMEN
CONTEXT: Barrett esophagus (BE) occurs in 1% to 10% of the general population and is believed to be the precursor of esophageal adenocarcinoma (EAC). The incidence of EAC has increased 350% in the last 3 decades without clear etiology. Finding predisposition genes may improve premorbid risk assessment, genetic counseling, and management. Genome-wide multiplatform approaches may lead to the identification of genes important in BE/EAC development. OBJECTIVE: To identify risk alleles or mutated genes associated with BE/EAC. DESIGN, SETTING, AND PATIENTS: Model-free linkage analyses of 21 concordant-affected sibling pairs with BE/EAC and 11 discordant sibling pairs (2005-2006). Significant germline genomic regions in independent prospectively accrued series of 176 white patients with BE/EAC and 200 ancestry-matched controls (2007-2010) were validated and fine mapped. Integrating data from these significant genomic regions with somatic gene expression data from 19 BE/EAC tissues yielded 12 "priority" candidate genes for mutation analysis (2010). Genes that showed mutations in cases but not in controls were further screened in an independent prospectively accrued validation series of 58 cases (2010). MAIN OUTCOME MEASURES: Identification of germline mutations in genes associated with BE/EAC cases. Functional interrogation of the most commonly mutated gene. RESULTS: Three major genes, MSR1, ASCC1, and CTHRC1 were associated with BE/EAC (all P < .001). In addition, 13 patients (11.2%) with BE/EAC carried germline mutations in MSR1, ASCC1, or CTHRC1. MSR1 was the most frequently mutated, with 8 of 116 (proportion, 0.069; 95% confidence interval [CI], 0.030-0.130; P < .001) cases with c.877C>T (p.R293X). An independent validation series confirmed germline MSR1 mutations in 2 of 58 cases (proportion, 0.035; 95% CI, 0.004-0.120; P = .09). MSR1 mutation resulted in CCND1 up-regulation in peripheral-protein lysate. Immunohistochemistry of BE tissues in MSR1-mutation carriers showed increased nuclear expression of CCND1. CONCLUSION: MSR1 was significantly associated with the presence of BE/EAC in derivation and validation samples, although it was only present in a small percentage of the cases.
Asunto(s)
Adenocarcinoma/genética , Esófago de Barrett/genética , Neoplasias Esofágicas/genética , Proteínas de la Matriz Extracelular/genética , Mutación de Línea Germinal , Receptores Depuradores de Clase A/genética , Factores de Transcripción/genética , Alelos , Proteínas de la Matriz Extracelular/metabolismo , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Receptores Depuradores de Clase A/metabolismo , HermanosRESUMEN
Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators of biological pathways. However, involvement of lncRNAs in the development of BE and EAC has not been well-studied. The aims of the current study were: (1) to study involvement of the lncRNA, miR205HG, in the development of BE and EAC; (2) to clarify the role of miR205HG in in vitro and in vivo experiments; and (3) to investigate the mechanism of miR205HG involving the Hedgehog (Hh) signaling pathway. These experiments revealed that miR205HG was downregulated in EAC vs. normal esophageal epithelia (NE) as well as in EAC cell lines, and its forced overexpression inhibited EAC cell proliferation and cell cycle progression in vitro. Similarly, overexpression of miR205HG inhibited xenograft tumor growth in mice In vivo. Finally, we show that one mechanism of action of miR205HG involves the Hh signaling pathway: miR205HG and Hh expression levels were inversely correlated in both EAC (r = -0.73) and BE (r = -0.83) tissues, and in vitro studies revealed details of Hh signaling inhibition induced by miR205HG. In conclusion, these findings establish that lncRNA miR205HG functions as a tumor suppressor in the development of BE and EAC, at least in part through its effect on the Hh signaling pathway.
RESUMEN
Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Antígenos de Neoplasias/biosíntesis , Antineoplásicos/farmacología , Línea Celular Tumoral , Metilación de ADN , Factor de Transcripción E2F1/metabolismo , Silenciador del Gen , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Modelos Biológicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , VorinostatRESUMEN
Translocation of chromosomes 8 and 21, t(8;21), resulting in the AML1-ETO fusion gene, is associated with acute myeloid leukemia. We searched for additional genomic abnormalities in this acute myeloid leukemia subtype by performing single nucleotide polymorphism genomic arrays (SNP-chip) analysis on 48 newly diagnosed cases. Thirty-two patients (67%) had a normal genome by SNP-chip analysis (Group A), and 16 patients (33%) had one or more genomic abnormalities including copy number changes or copy number neutral loss of heterozygosity (Group B). Two samples had copy number neutral loss of heterozygosity on chromosome 6p including the PIM1 gene; and one of these cases had E135K mutation of Pim1. Interestingly, 38% of Group B and only 13% of Group A samples had a KIT-D816 mutation, suggesting that genomic alterations are often associated with a KIT-D816 mutation. Importantly, prognostic analysis revealed that overall survival and event-free survival of individuals in Group B were significantly worse than those in Group A.
Asunto(s)
Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Genoma Humano , Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleótido Simple , Translocación Genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Supervivencia sin Enfermedad , Humanos , Leucemia Mieloide Aguda/mortalidad , Pérdida de Heterocigocidad/genética , Mutación Missense , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-pim-1/genética , Proteína 1 Compañera de Translocación de RUNX1 , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The FBXW7 (also known as AGO, hCDC4, FBW7 and SEL-10) gene encodes a subunit of an ubiquitin protein ligase which regulates levels of cyclin E, NOTCH and other proteins. Engineered FBXW7 null cells display cell cycle and chromosome stability defects. Mutations of FBXW7 have been found in human colorectal, ovarian, endometrial tumors and T-cell acute lymphocytic leukemias. Prompted by these findings we have examined acute myeloid leukemia, non-Hodgkin's lymphoma, T-cell acute lymphocytic leukemia, B-cell acute lymphocytic leukemia and adult T-cell leukemia DNA for mutations of the FBXW7 gene. Mutations were detected by PCR-SSCP of all coding exons of the three isoforms of FBXW7, shifted bands were direct sequenced. As expected, mutations were found in T-cell acute lymphocytic leukemias. However mutations of FBXW7 were also found in four of 118 B-cell acute lymphocytic leukemias and one of 24 adult T-cell leukemia samples. The nucleotide changes consisted of an insertion, resulting in a frameshift mutation, and missense mutations of highly conserved residues. All mutations affected the FBXW7 target interacting domain. These observations suggest that disruption of FBXW7 has a role in several forms of lymphocytic leukemias and not exclusively T-cell acute lymphocytic leukemia.
Asunto(s)
Linfoma de Burkitt/genética , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Leucemia-Linfoma de Células T del Adulto/genética , Linfoma no Hodgkin/genética , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Neoplasias de la Próstata/genética , Sarcoma/genética , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/genéticaRESUMEN
Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.
Asunto(s)
Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Humanos , Células MCF-7RESUMEN
There have been no reports describing the effects of cancer cell-derived extracellular vesicles (EVs) on three-dimensional organoids. In this study, we delineated the proneoplastic effects of esophageal adenocarcinoma (EAC)-derived EVs on gastric organoids (gastroids) and elucidated molecular mechanisms underlying these effects. EVs were identified using PKH-67 staining. Morphologic changes, Ki-67 immunochemistry, cell viability, growth rates, and expression levels of miR-25 and miR-210, as well as of their target mRNAs, were determined in gastroids co-cultured with EAC-derived extracellular vesicles (c-EVs). C-EVs were efficiently taken up by gastroids. Notably, c-EV-treated gastroids were more crowded, compact, and multilayered and contained smaller lumens than did those cultured in organoid medium alone or in EAC-conditioned medium that had been depleted of EVs. Moreover, c-EV-treated gastroids manifested increased proliferation and cellular viability relative to medium-only or EV-depleted controls. Expression levels of miR-25 and miR-210 were significantly higher, and those of PTEN and AIFM3 significantly lower, in c-EV-treated versus medium-only or EV-depleted control groups. Inhibitors of miR-25 and miR-210 reversed the increased cell proliferation induced by c-exosomes in co-cultured gastroids by lowering miR-25 and miR-210 levels. In conclusion, we have constructed a novel model system featuring the co-culture of c-EVs with three-dimensional gastroids. Using this model, we discovered that cancer-derived EVs induce a neoplastic phenotype in gastroids. These changes are due, at least in part, to EV transfer of miR-25 and miR-210.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Vesículas Extracelulares/metabolismo , Mucosa Gástrica/metabolismo , MicroARNs/metabolismo , Organoides/metabolismo , Fenotipo , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Transformación Celular Neoplásica , Técnicas de Cocultivo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Mucosa Gástrica/patología , Humanos , MicroARNs/administración & dosificación , MicroARNs/genética , Organoides/patologíaRESUMEN
Thymoquinone (TQ) has been reported to possess anti-tumor activity in various types of cancer. However, its effects and molecular mechanism of action in hepatocellular carcinoma (HCC) are still not completely understood. We observed that TQ inhibited tumor cell growth in vitro, where treatment with TQ arrested the cell cycle in G1 by upregulating p21 and downregulating cyclinD1 and CDK2 expression; moreover, TQ induced apoptosis by decreasing expression of Bcl-2 and increasing expression of Bax. Simultaneously, TQ demonstrated a suppressive impact on the Notch pathway, where overexpression of NICD1 reversed the inhibitory effect of TQ on cell proliferation, thereby attenuating the repressive effects of TQ on the Notch pathway, cyclinD1, CDK2 and Bcl-2, and also diminishing upregulation of p21 and Bax. In a xenograft model, TQ inhibited HCC growth in nude mice; this inhibitory effect in vivo, as well as of HCC cell growth in vitro, was associated with a discernible decline in NICD1 and Bcl-2 levels and a dramatic rise in p21 expression. In conclusion, TQ inhibits HCC cell growth by inducing cell cycle arrest and apoptosis, achieving these effects by repression of the Notch signaling pathway, suggesting that TQ represents a potential preventive or therapeutic agent in HCC patients.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Benzoquinonas/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Receptor Notch1/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated C/EBPß and C/EBPε double-knockout (bbee) mice and compared their phenotypes to those of single deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2-3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(-)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of LPS- and IFNγ-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct C/EBP-targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Neutrófilos/metabolismo , Animales , Animales Recién Nacidos , Infecciones Bacterianas/embriología , Infecciones Bacterianas/genética , Infecciones Bacterianas/microbiología , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/deficiencia , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/deficiencia , Proteínas Potenciadoras de Unión a CCAAT/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Neutrófilos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Asparagine synthetase (ASNS) is an enzyme expressed ubiquitously in mammalian cells. Here, we discovered two 14-bp tandem repeat (2R, wild-type) sequences in the first intron of the gene. The 14-bp sequence is similar to the three GC-boxes (GC-I, -II, and -III) found in the promoter region of the ASNS gene, as well as, the binding site of transcription factor Sp-1. Approximately 75% of acute lymphoblastic leukemia (ALL) samples had the 2R sequence in both allele; however, 20% and 3% ALL samples had three (3R) and four (4R) 14-bp tandem repeats in one allele, respectively; the other allele had 2R. The tandem repeat sequence was not specific to the leukemia cells but represents a novel germline polymorphism. Interestingly, the 14-bp sequence functioned as a transcriptional enhancer element as shown by reporter analysis and formed a protein-DNA complex in vitro. Our data for the first time show that the ASNS gene has tandem repeated sequences as a polymorphism, and it can function as a transcriptional element; increased number of tandem repeat producing increased activity. Clinical significance in ALL requires further studies.
Asunto(s)
Aspartatoamoníaco Ligasa/genética , ADN de Neoplasias/genética , Regulación Enzimológica de la Expresión Génica , Polimorfismo Genético/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Secuencias Repetidas en Tándem/genética , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Humanos , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Pancreatic cancer is an aggressive malignancy that is generally refractory to chemotherapy, thus posing experimental and clinical challenges. In this study, the antiproliferative effect of the triterpenoid compound cucurbitacin B was tested in vitro and in vivo against human pancreatic cancer cells. Dose-response studies showed that the drug inhibited 50% growth of seven pancreatic cancer cell lines at 10(-7) mol/L, whereas clonogenic growth was significantly inhibited at 5 x 10(-8) mol/L. Cucurbitacin B caused dose- and time-dependent G(2)-M-phase arrest and apoptosis of pancreatic cancer cells. This was associated with inhibition of activated JAK2, STAT3, and STAT5, increased level of p21(WAF1) even in cells with nonfunctional p53, and decrease of expression of cyclin A, cyclin B1, and Bcl-XL with subsequent activation of the caspase cascade. Interestingly, the combination of cucurbitacin B and gemcitabine synergistically potentiated the antiproliferative effects of gemcitabine on pancreatic cancer cells. Moreover, cucurbitacin B decreased the volume of pancreatic tumor xenografts in athymic nude mice by 69.2% (P < 0.01) compared with controls without noticeable drug toxicities. In vivo activation of JAK2/STAT3 was inhibited and expression of Bcl-XL was decreased, whereas caspase-3 and caspase-9 were up-regulated in tumors of drug-treated mice. In conclusion, we showed for the first time that cucurbitacin B has profound in vitro and in vivo antiproliferative effects against human pancreatic cancer cells, and the compound may potentate the antiproliferative effect of the chemotherapeutic agent gemcitabine. Further clinical studies are necessary to confirm our findings in patients with pancreatic cancer.