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1.
Biol Res ; 53(1): 14, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293550

RESUMO

BACKGROUND: Previous studies have shown that long noncoding RNA (lncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this lncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR-490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.


Assuntos
MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose , Carcinogênese/metabolismo , Linhagem Celular Tumoral/metabolismo , Movimento Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos BALB C , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Biol Res ; 53(1): 13, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293552

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.


Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Neoplasias da Vesícula Biliar/genética , Índios Sul-Americanos/genética , Animais , Antineoplásicos/farmacologia , Líquido Ascítico/metabolismo , Carcinogênese/genética , Testes de Carcinogenicidade , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Chile , Cisplatino/farmacologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Impressões Digitais de DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Células Epiteliais/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica , Genes erbB-2/genética , Humanos , Queratina-19/genética , Queratina-7/genética , Masculino , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Análise de Sequência de RNA , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
3.
J Cardiothorac Surg ; 15(1): 18, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931858

RESUMO

INTRODUCTION: Lung cancer is the leading causes of cancer-related deaths globally. The most frequent histologic type of lung cancer is non-small-cell lung cancer (NSCLC). NSCLC often undergo epithelial-mesenchymal transition (EMT). The components that control this process are thus promising therapeutic targets. MATERIALS AND METHODS: Gli/EMT protein expression levels were examined by western blot in paired NSCLC patient tissues and NSCLC cell lines. Functional analyses were performed to investigate SHH/Gli signaling and EMT in NSCLC cell lines. MTS cell viability, luciferase reporter, and western blot assays were performed to analyze pathway activity, while wound healing and transwell assays were executed to measure cell migration and invasion. RESULTS: Higher Gli1 expressions were detected in tumor samples than in paired normal tissues. Differential expression of EMT biomarkers and activation of p-AKT were observed in tumor tissues. N-Shh stimulation of cells significantly increased reporter activity in NSCLC cell lines, while Gli-i treatment of transfected cells showed less relative reporter activity. When subjected to both Gli-i and N-Shh treatment, NSCLC cell lines continued to demonstrate decreased Gli transcriptional activity. Gli inhibition is associated with decreased expression level of p-AKT, N-cadherin and Vimentin. Knockdown of both Gli1 and Gli2 showed decreased EMT, migrative and invasive ability. Cells stimulated by N-Shh demonstrated greater mobility. In addition, AKT-i treated cells also demonstrated inhibited EMT activity. CONCLUSIONS: This study provides evidence for aberrant upregulation of the Gli signaling pathway and a strong association between expression of Gli versus AKT and EMT markers in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pulmonares/metabolismo , Proteína GLI1 em Dedos de Zinco/fisiologia , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral/metabolismo , Movimento Celular/efeitos dos fármacos , Proteínas Hedgehog/fisiologia , Humanos , Transdução de Sinais/fisiologia , Proteína GLI1 em Dedos de Zinco/metabolismo
4.
Mol Cell ; 76(5): 838-851.e5, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31564558

RESUMO

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/metabolismo , Metaboloma/fisiologia , Biomarcadores Tumorais/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Neoplasias/metabolismo
5.
BMC Vet Res ; 15(1): 357, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640712

RESUMO

BACKGROUND: Canine and human osteosarcomas (OS) are notably similar and have a high rate of metastasis. There is a poor understanding of the tumor development process, predisposing causes, and varying levels of aggression among different cell lines. By characterizing newly developed canine osteosarcoma cell lines, treatments for people and pets can be developed. Of the seven subtypes of OS, three are represented in this group: osteoblastic (the most common), fibroblastic, and giant cell variant. To our knowledge, there are no other giant cell variant canine OS cell lines in the published literature and only one canine fibroblastic osteosarcoma cell line. Understanding the differences between the histologic subtypes in dogs will help to guide comparative research. RESULTS: Alkaline phosphatase expression was ubiquitous in all cell lines tested and invasiveness was variable between the cell lines tested. Invasiveness and oxidative damage were not correlated with in vivo growth rates, where TOT grew the fastest and had the higher percentage of mice with metastatic lesions. TOL was determined to be the most chemo-resistant during cisplatin chemotherapy while TOM was the most chemo-sensitive. CONCLUSIONS: Further comparisons and studies using these cell lines may identify a variety of characteristics valuable for understanding the disease process and developing treatments for osteosarcoma in both species. Some of this data was presented as a poster by KMF at the August 5th, 2017 National Veterinary Scholars Program in Bethesda, MA. Characterization of 5 newly generated canine osteosarcoma cell lines. Kelli Franks, Tasha Miller, Heather Wilson-Robles.


Assuntos
Linhagem Celular Tumoral , Doenças do Cão/metabolismo , Osteossarcoma/veterinária , Adipogenia , Fosfatase Alcalina/biossíntese , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Condrogênese , Cisplatino/farmacologia , Meios de Cultura , Doenças do Cão/patologia , Cães , Feminino , Xenoenxertos/metabolismo , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Osteogênese , Osteossarcoma/metabolismo
6.
Nat Commun ; 10(1): 3838, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444335

RESUMO

Developing biomimetic nanoparticles without loss of the integrity of proteins remains a major challenge in cancer chemotherapy. Here, we develop a biocompatible tumor-cell-exocytosed exosome-biomimetic porous silicon nanoparticles (PSiNPs) as drug carrier for targeted cancer chemotherapy. Exosome-sheathed doxorubicin-loaded PSiNPs (DOX@E-PSiNPs), generated by exocytosis of the endocytosed DOX-loaded PSiNPs from tumor cells, exhibit enhanced tumor accumulation, extravasation from blood vessels and penetration into deep tumor parenchyma following intravenous administration. In addition, DOX@E-PSiNPs, regardless of their origin, possess significant cellular uptake and cytotoxicity in both bulk cancer cells and cancer stem cells (CSCs). These properties endow DOX@E-PSiNPs with great in vivo enrichment in total tumor cells and side population cells with features of CSCs, resulting in anticancer activity and CSCs reduction in subcutaneous, orthotopic and metastatic tumor models. These results provide a proof-of-concept for the use of exosome-biomimetic nanoparticles exocytosed from tumor cells as a promising drug carrier for efficient cancer chemotherapy.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Exossomos/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Exocitose , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/química , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Porosidade , Estudo de Prova de Conceito , Silício/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Toxicol Lett ; 315: 77-86, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31470059

RESUMO

T-2 toxin is a major pollutant in crops and feedstuffs. Due to its high toxicity in a variety of organisms, T-2 toxin is of great concern as a threat to humans and to animal breeding. Overexpression of CYP1A1 may contribute to carcinogenesis, and CYP1A1 may be a promising target for the prevention and treatment of human malignancies. Therefore, it is essential to understand the regulatory mechanism by which T-2 toxin induces CYP1A1 expression in human cells. In this study, we confirmed that T-2 toxin (100 ng/mL) induced the expression of CYP1A1 in HepG2 cells through NRF1 and Sp1 bound to the promoter instead of through the well-recognized Aromatic hydrocarbon receptors (AhR). In cells treated with T-2 toxin, Sp1, but not NRF1, was significantly upregulated. However, T-2 toxin apparently promoted the interaction between NRF1 and Sp1 proteins, as revealed by IP analysis. Furthermore, in T-2 toxin-treated HepG2 cells, nuclear translocation of NRF1 was enhanced, while knockdown of Sp1 ablated NRF1 nuclear enrichment. Our results revealed that the upregulation of CYP1A1 by T-2 toxin in HepG2 cells depended on enhanced interaction between Sp1 and NRF1. This finding suggests the tumorigenic features of T-2 toxin might be related to the CYP1A1, which provides new insights to understand the toxicological effect of T-2 toxin.


Assuntos
Citocromo P-450 CYP1A1/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 1 Relacionado a NF-E2/genética , Fator de Transcrição Sp1/genética , Toxina T-2/toxicidade , Regulação para Cima/efeitos dos fármacos , Carcinoma/fisiopatologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Pesquisas com Embriões , Regulação Enzimológica da Expressão Gênica , Humanos , Rim , Neoplasias Hepáticas/fisiopatologia , Fator 1 Relacionado a NF-E2/efeitos dos fármacos , Fator 1 Relacionado a NF-E2/metabolismo , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo
8.
Genes Cells ; 24(10): 674-681, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433897

RESUMO

Forkhead box (FOX) proteins constitute a family of transcription factors that are evolutionarily conserved in various species ranging from yeast to humans. These proteins have functions during development as well as in adulthood. To date, many reports have described the functions of FOX family genes in cancer cells, but the role of FOXB2 is not well understood. In one of the pancreas ductal adenocarcinoma cell lines, Panc-1 cells, we showed here that FOXB2 expression is barely detectable and that CpG islands in the 5' regions of the FOXB2 are highly methylated. These findings led us to hypothesize that FOXB2 acts as a tumor suppressor. To clarify our hypotheses, we investigated the effects of FOXB2 over-expression in Panc-1 cells. We obtained FOXB2 stable transfectants, and these clones exhibited reduced spheroid formation ability. Expression of ß-catenin, which is reported to be over-expressed in various cancer cells, was highly suppressed in FOXB2 stable transfectants. Moreover, side population (SP) cell fractions, which have a high tumorigenesis and metastatic potential, as well as anchorage-independent growth ability, were reduced. These results suggest that FOXB2 has the ability to inhibit the malignant characteristics of Panc-1 in vitro.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/genética , Fatores de Transcrição Forkhead/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética
9.
BMC Biol ; 17(1): 37, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039782

RESUMO

BACKGROUND: Cancer cells reprogram their metabolism to survive and propagate. Thus, targeting metabolic rewiring in tumors is a promising therapeutic strategy. Genome-wide RNAi and CRISPR screens are powerful tools for identifying genes essential for cancer cell proliferation and survival. Integrating loss-of-function genetic screens with genomics and transcriptomics datasets reveals molecular mechanisms that underlie cancer cell dependence on specific genes; though explaining cell line-specific essentiality of metabolic genes was recently shown to be especially challenging. RESULTS: We find that variability in tissue culture medium between cell lines in a genetic screen is a major confounding factor affecting cell line-specific essentiality of metabolic genes-while, quite surprisingly, not being previously accounted for. Additionally, we find that altered expression level of a metabolic gene in a certain cell line is less indicative of its essentiality than for other genes. However, cell line-specific essentiality of metabolic genes is significantly correlated with changes in the expression of neighboring enzymes in the metabolic network. Utilizing a machine learning method that accounts for tissue culture media and functional association between neighboring enzymes, we generated predictive models for cancer cell line-specific dependence on 162 metabolic genes (representing a ~ 2.2-fold increase compared to previous studies). The generated predictive models reveal numerous novel associations between molecular features and cell line-specific dependency on metabolic genes. Specifically, we demonstrate how cancer cell dependence on one-carbon metabolic enzymes is explained based on cancer lineage, oncogenic mutations, and RNA expression of neighboring enzymes. CONCLUSIONS: Considering culture media as well as accounting for molecular features of functionally related metabolic enzymes in a metabolic network significantly improves our understanding of cancer cell line-specific dependence on metabolic genes. We expect our approach and predictive models of metabolic gene essentiality to be a useful tool for investigating metabolic abnormalities in cancer.


Assuntos
Linhagem Celular Tumoral/metabolismo , Testes Genéticos , Neoplasias/genética , Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Interferência de RNA
10.
J Biotechnol ; 301: 79-87, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31145935

RESUMO

Nuclear Factor kappa B (NF-κB) is a conserved transcription factor involved in the expression of genes that are critical to inflammation and cell survival. Exposure to particular signals results in phosphorylation of NF-κB inhibitor proteins, which in turn allows NF-κB dimers to translocate to the nucleus and induce gene expression. Pathologic consequences of NF-κB activation are vast, mainly because of the pleiotropic roles that NF-κB-induced genes have on inflammation, cell proliferation and apoptosis. Thus, experimental models assessing NF-κB activation have direct screening applications for drug discovery. In this scenario, pathway-specific reporter cell systems become valuable tools to identify and elucidate the mechanism of action of novel compounds. Here, we describe the generation, characterization, and validation of human cancer epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory mediators. Human lung (H460) and breast (T-47D) cancer cell lines were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. The pro-inflammatory cytokine TNF-α was able to activate the reporter systems in a concentration-response manner, correlating to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using dexamethasone, a potent anti-inflammatory drug, a synthetic inhibitor of NF-κB (BAY 11-7082) and a new anti-cancer peptide (CIGB-552). We have established robust H460-NF-κB-hrGFP and T-47D-NF-κB-hrGFP reporter cell lines which represent a useful cancer model for primary screening and identification of compounds with anti-inflammatory activity.


Assuntos
Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/metabolismo , Descoberta de Drogas/métodos , Neoplasias Pulmonares/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Feminino , Humanos , Modelos Biológicos , Reprodutibilidade dos Testes
11.
Arch Endocrinol Metab ; 63(2): 142-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30916164

RESUMO

OBJECTIVE: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway. MATERIALS AND METHODS: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. RESULTS: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. CONCLUSION: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.


Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Fator de Crescimento Transformador alfa/genética , Tri-Iodotironina/genética , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/metabolismo , Feminino , Humanos , Células MCF-7/metabolismo , Proto-Oncogenes/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia
12.
Nat Commun ; 10(1): 991, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824700

RESUMO

6-Phosphogluconate dehydrogenase (6PGD) is a key enzyme that converts 6-phosphogluconate into ribulose-5-phosphate with NADP+ as cofactor in the pentose phosphate pathway (PPP). 6PGD is commonly upregulated and plays important roles in many human cancers, while the mechanism underlying such roles of 6PGD remains elusive. Here we show that upon EGFR activation, 6PGD is phosphorylated at tyrosine (Y) 481 by Src family kinase Fyn. This phosphorylation enhances 6PGD activity by increasing its binding affinity to NADP+ and therefore activates the PPP for NADPH and ribose-5-phosphate, which consequently detoxifies intracellular reactive oxygen species (ROS) and accelerates DNA synthesis. Abrogating 6PGD Y481 phosphorylation (pY481) dramatically attenuates EGF-promoted glioma cell proliferation, tumor growth and resistance to ionizing radiation. In addition, 6PGD pY481 is associated with Fyn expression, the malignancy and prognosis of human glioblastoma. These findings establish a critical role of Fyn-dependent 6PGD phosphorylation in EGF-promoted tumor growth and radiation resistance.


Assuntos
Neoplasias/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/efeitos da radiação , Proliferação de Células , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Células HEK293 , Humanos , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , NADP/metabolismo , Neoplasias/patologia , Via de Pentose Fosfato , Fosforilação , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Ribosemonofosfatos/metabolismo , Regulação para Cima
13.
Saudi J Gastroenterol ; 25(2): 119-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30719998

RESUMO

Background/Aims: This study aimed to explore the effect of circular RNA ARHGAP26 (circ-ARHGAP26) on cell proliferation and apoptosis in gastric cancer (GC) cell lines. Materials and Methods: Human GC cell lines including HGC-27, AGS, SGC-7901, BGC-823, NCI-N87 and human normal gastric mucosal cells GSE-1 were cultured. The circ-ARHGAP26 expression was determined by quantitative polymerase chain reaction assay. Blank inhibitor and circ-ARHGAP26 inhibitor plasmids were transfected into HGC-27 or AGS cells as NC (-) and circ-ARHGAP26(-) groups. Counting Kit-8 (CCK-8) and Annexin V (AV)/propidium iodide (PI) were conducted to evaluate cell proliferation and cell apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic markers (C-Caspase3 and Bcl-2). Results: The circ-ARHGAP26 expression was elevated in HGC-27 (P < 0.001), AGS (P < 0.001), SGC-7901 (P < 0.01), BGC-823 (P < 0.05) and NCI-N87 (P < 0.05) GC cell lines compared to GSE-1 cells. In HGC-27 cells, CCK8 assay revealed that cell proliferation was decreased at 48 h (P < 0.05) and 72 h (P < 0.01), while AV/PI assay disclosed that cell apoptosis rate was increased at 72 h in circ-ARHGAP26 (-) group compared to NC (-) group (P < 0.01). Western blot assay also illuminated that apoptotic marker C-Caspase 3 was raised, while anti-apoptotic marker Bcl-2 was reduced at 72 h in circ-ARHGAP26 (-) group compared to NC (-) group. In addition, further validation in AGS cells also exhibited that cells proliferation was repressed, while apoptosis was enhanced in circ-ARHGAP26 (-) group compared to NC (-) group. Conclusion: The circ-ARHGAP26 is over-expressed and its downregulation inhibits cell proliferation and promotes cells apoptosis in GC cells.


Assuntos
Apoptose/genética , Proliferação de Células/genética , RNA/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral/metabolismo , Regulação para Baixo , Proteínas Ativadoras de GTPase/metabolismo , Mucosa Gástrica/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Gástricas/patologia
14.
Environ Pollut ; 246: 763-771, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30623832

RESUMO

Ambient ultrafine black carbon (uBC) can potentially cross blood-brain barrier, however, very little is currently known about the effects they may have on central nervous system. This study aimed to explore the roles of autophagy in Alzheimer-like pathogenic changes promoted by uBC in SH-SY5Y cells. We firstly found uBC could cause cytotoxicity and oxidative stress in SH-SY5Y cells. Additionally we found uBC initiated progressive development of Alzheimer's disease (AD) associated features, mainly including neuro-inflammation and phosphorylation of tau protein (p-Tau) accumulation. Meanwhile, autophagy process was activated by uBC probably through phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway. RNA interference and autophagosome-lysosome fusion inhibitor were applied to block autophagy process at different stages. Autophagy dysfunction at the initial membrane expansion stage could aggravate p-Tau accumulation and other Alzheimer-like changes in SH-SY5Y cells promoted by uBC. However, autophagy inhibition at the final stage could alleviate p-Tau accumulation caused by uBC. This suggested that inhibition of the infusion of autophagosome and lysosome could possibly activate ubiquitination degradation pathway to regulate p-Tau equilibrium in SH-SY5Y cells. Our findings further raise the concerns about the effects of uBC on the risk of AD and indicate potential roles of autophagy in early Alzheimer-like pathogenic changes caused by ambient uBC.


Assuntos
Doença de Alzheimer/metabolismo , Autofagia/efeitos dos fármacos , Carbono/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Neuroblastoma/metabolismo , Humanos
15.
Med Sci Monit ; 25: 500-515, 2019 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-30653481

RESUMO

BACKGROUND TCL-based immunotherapy has been applied in the field cancer therapy. However, it is un clear whether this therapy can be used to treat triple-negative breast cancer (TNBC), and different TNBC cells have distinct responses to this therapy. MATERIAL AND METHODS In the present work, we conducted 2 different TCL-based immunotherapies to treat TNBC and compared their anti-tumor effect on 4 TNBC cell lines: MDA-MB-231, MDA-MB-436, HCC1937, and HCC1187. RESULTS Peripheral blood mononuclear cells (PBMC) activated by TCL and peripheral blood lymphocytes (PBL) stimulated with TCL-loaded DC demonstrated the ability to kill TNBC cells in vitro, but the killing efficiency of PBL was much higher than that of PBMC. In vivo, PBL stimulated with TCL-loaded DC can also stop the growth of TNBC tumors in mice. HCC1187 and MDA-MD-231 best respond to TCL-based immunotherapy both in vitro and in vivo. The response of HCC1937 was weaker, and that of MDA-MB-436 was lowest among the 4 cell lines. Total mRNA microarray analysis of TNBC cells showed that PDL-1 mRNA expression in HCC1937 and MDA-MD-436 cells was higher than in the other 2 TNBC cell lines, and that of MDA-MB-436 was higher than that of HCC1937. PD1 blocking can decrease the apoptosis rate. These results show that different contents of PDL-1 in TCL, by interacting with PD expression on lymphocytes, can induce different ratios of lymphocyte apoptosis, and then result in distinct response of the 4 TNBC cell lines to TCL-based immunotherapy. CONCLUSIONS TCL-based immunotherapy has discrepant anti-tumor efficiency in different TNBC cell lines by PDL-1/PD interaction, providing the theoretical basis of TCL-based immunotherapy in TNBC.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Feminino , Humanos , Imunoterapia , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Nus , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Exp Hematol ; 69: 27-36, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352278

RESUMO

Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.


Assuntos
Antígenos CD34/biossíntese , Linhagem Celular Tumoral , Deleção Cromossômica , Inversão Cromossômica , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 7/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda , Proteínas de Neoplasias/biossíntese , Adulto , Antígenos CD34/genética , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Cromossomos Humanos Par 3/metabolismo , Cromossomos Humanos Par 7/metabolismo , Técnicas de Cocultura , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Células Estromais/metabolismo , Células Estromais/patologia
17.
JCI Insight ; 3(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385715

RESUMO

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , c-Mer Tirosina Quinase/metabolismo , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Humanos , Imunoterapia/métodos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptor de Morte Celular Programada 1/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , c-Mer Tirosina Quinase/antagonistas & inibidores
18.
Mol Biol Rep ; 45(6): 2689-2695, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30390187

RESUMO

Cancer cell lines are used worldwide in biomedical researches, and data interpretation solely depends on unambiguous attribution of those respective cell lines to its original sources. Approximately one-third of all cell lines have an origin other than that assumed, leading to invalid results. It is necessary to characterize the origin of cell lines. Short-tandem-repeat (STR) fingerprinting (DNA fingerprinting) is the method for characterization of genetic identity in cultured cell lines under certain experimental conditions. We showed the fingerprinting profiles in a summed and unidentified human cancer cell line comparison to HCC1954 cell line, revealing marked alterations in DNA fingerprinting profiles up to fourteen STR loci from 16 loci. Furthermore, Sanger DNA sequencing showed no c.3140A > G heterozygous mutation in the PIK3CA gene of this suspected HCC1954 cell line. In addition, we showed the fingerprinting profiles in an unidentified cancer cell line comparison to SiHa cervical cell line, revealing same DNA fingerprinting profiles. In conclusion, we have successfully authenticated and identified both suspected HCC1954 and SiHa cell lines by STR analysis and DNA sequencing. STR analysis combined DNA sequencing may be very useful to evaluate genotypes of cancer cell lines in our cancer studies, as well as in judicial authentication and forensic sciences.


Assuntos
Linhagem Celular Tumoral/classificação , Impressões Digitais de DNA/métodos , Repetições de Microssatélites/genética , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/fisiologia , Genótipo , Humanos , Controle de Qualidade , Análise de Sequência de DNA/métodos
19.
Medicina (Kaunas) ; 54(2)2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30344242

RESUMO

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Perfilação da Expressão Gênica/métodos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Meios de Cultivo Condicionados/química , Feminino , Humanos , Células MCF-7/efeitos dos fármacos , Células MCF-7/metabolismo , Células MCF-7/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Sci Rep ; 8(1): 14654, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279592

RESUMO

Lonidamine (LND), a metabolic modulator, sensitizes DB-1 human melanoma to doxorubicin (DOX) chemotherapy by acidifying and de-energizing the tumor. This report compares the effects of LND on two human melanoma lines, DB-1 and WM983B, which exhibit different metabolic properties. Using liquid chromatography mass spectrometry and Seahorse analysis, we show that DB-1 was more glycolytic than WM983B in vitro. 31P magnetic resonance spectroscopy (MRS) indicates that LND (100 mg/kg, i.p.) induces similar selective acidification and de-energization of WM983B xenografts in immunosuppressed mice. Over three hours, intracellular pH (pHi) of WM983B decreased from 6.91 ± 0.03 to 6.59 ± 0.10 (p = 0.03), whereas extracellular pH (pHe) of this tumor changed from 7.03 ± 0.05 to 6.89 ± 0.06 (p = 0.19). A decline in bioenergetics (ß-NTP/Pi) of 55 ± 5.0% (p = 0.03) accompanied the decline in pHi of WM983B. Using 1H MRS with a selective multiquantum pulse sequence and Hadamard localization, we show that LND induced a significant increase in tumor lactate levels (p < 0.01). LND pre-treatment followed by DOX (10 mg/kg, i.v.) produced a growth delay of 13.7 days in WM983B (p < 0.01 versus control), a growth delay significantly smaller than the 25.4 days that occurred with DB-1 (p = 0.03 versus WM983B). Differences in relative levels of glycolysis may produce differential therapeutic responses of DB-1 and WM983B melanomas.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Doxorrubicina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Indazóis/farmacologia , Melanoma/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Doxorrubicina/uso terapêutico , Sinergismo Farmacológico , Glucose/análise , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Indazóis/uso terapêutico , Ácido Láctico/análise , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Melanoma/patologia , Camundongos , Camundongos Nus , Oxigênio/análise , Oxigênio/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
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