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1.
J Pers Med ; 12(5)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35629089

RESUMO

The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.

2.
Oncol Lett ; 16(1): 809-814, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29963148

RESUMO

Oesophageal squamous cell carcinoma (ESCC) is an aggressive cancer that resulted in ~400,000 mortalities worldwide in 2012. It was reported previously that fibroblast growth factor receptor-like 1 (FGFRL1) is highly expressed in ESCC patients with lymph node metastasis and poor prognosis accordingly. FGFRL1 is an FGFR that lacks tyrosine kinase activity, whereas the activity is critical for other FGFRs to activate intracellular signalling. The mechanism by which FGFRL1 promotes the aggressiveness of ESCCs is unknown. In the present study, two independent FGFRL1-deficient cell lines were generated from human ESCC KYSE520 cells, in order to investigate the relationship of FGFRL1 with the aggressiveness of ESCCs. FGFRL1-deficiency did not affect proliferation of KYSE520 cells in vitro. However, a xenograft mouse model demonstrated that FGFRL1-deficiency decelerated tumour growth in vivo. The haematoxylin-eosin staining identified that FGFRL1-deficient cells formed well-differentiated squamous cell carcinomas, whereas wild-type cells formed moderately differentiated squamous cell carcinomas. Microarray analysis of mRNA expression revealed that FGFRL1-depletion resulted in decreased expression of proteins associated with motility and invasion of tumour cells, matrix metalloproteinase-1 and fibroblast growth factor binding protein 1. The wound-healing assay indicated that depleting FGFRL1 reduced cell motility. Furthermore, the invasiveness of FGFRL1-deficient cells was lesser than that of wild-type KYSE520 cells. In the FGFRL1-deficient KYSE520 cells, actin filaments around the nucleus were observed sparsely, whereas the filaments along the plasma membranes were observed as frequently as those in the parent KYSE520 cells. These results demonstrate that FGFRL1 may be involved in regulation of protein expression, actin filament assembly and tumorigenic potential of ESCC cells.

3.
Biomed Res Int ; 2015: 960840, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26171396

RESUMO

The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.


Assuntos
Neoplasias da Mama/metabolismo , Técnicas de Cocultura/métodos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Proteínas/análise , Proteínas/genética , Proteínas/metabolismo , Transcriptoma/genética , Regulação para Cima
4.
Int J Oncol ; 44(6): 1923-32, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24692008

RESUMO

MicroRNA (miR)-203 has been shown to induce squamous differentiation of epidermal stem cells through the suppression of p63. The aim of this study was to assess the tumor suppressor effect of miR-203 in esophageal squamous cell carcinoma (ESCC) with focus on the regulation of the cell fate decisions and organization of tumor tissue architecture in vivo. Our investigation establishing stable clones from ESCC cell lines with induced miR-203 expression resulted in significant growth inhibition in a mouse xenograft model. Small foci were observed in xenograft tumors with stratified squamous differentiation in conjunction with restored baso-apical polarity. The expression of the basement membrane protein laminine was localized at the center of the foci and the basal cell marker p75NTR was expressed in the innermost layer. The expression of ki67 and p63 was co-localized at the center layers, while involucrin was expressed in the outer layers. Flow cytometry revealed that the p75NTR-positive cells expressing p63 and Bmi1 were well maintained, while the expression of p63 was suppressed in the p75NTR-negative cells. Our cDNA microarray analysis demonstrated the upregulation of genes involved in regulating tissue architecture, such as BMP-4 and ZO-1 in the mir-203 transfectant. Investigation using surgically removed ESCC specimens revealed that the expression of miR-203 significantly correlated with a favorable prognosis. These results demonstrated that miR-203 regulated both basal and supra-basal cell components to induce differentiation with restored epithelial tissue architecture, leading to significant tumor growth inhibition in vivo. Those results suggest the use of miR-203 as a novel therapeutic and diagnostic target in patients with ESCC.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Neoplasia de Células Basais/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/cirurgia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/cirurgia , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasia de Células Basais/metabolismo , Neoplasia de Células Basais/cirurgia , Neoplasias Experimentais , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Cancer ; 134(5): 1067-76, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23959426

RESUMO

Activation-induced cytidine deaminase (AID) contributes to inflammation-associated carcinogenesis through its mutagenic activity. In our study, by taking advantage of the ability of AID to induce genetic aberrations, we investigated whether liver cancer originates from hepatic stem/progenitor cells that accumulate stepwise genetic alterations. For this purpose, hepatic progenitor cells enriched from the fetal liver of AID transgenic (Tg) mice were transplanted into recipient "toxin-receptor mediated conditional cell knockout" (TRECK) mice, which have enhanced liver regeneration activity under the condition of diphtheria toxin treatment. Whole exome sequencing was used to determine the landscape of the accumulated genetic alterations in the transplanted progenitor cells during tumorigenesis. Liver tumors developed in 7 of 11 (63.6%) recipient TRECK mice receiving enriched hepatic progenitor cells from AID Tg mice, while no tumorigenesis was observed in TRECK mice receiving hepatic progenitor cells of wild-type mice. Histologic examination revealed that the tumors showed characteristics of hepatocellular carcinoma and partial features of cholangiocarcinoma with expression of the AID transgene. Whole exome sequencing revealed that several dozen genes acquired single nucleotide variants in tumor tissues originating from the transplanted hepatic progenitor cells of AID Tg mice. Microarray analyses revealed that the majority of the mutations (>80%) were present in actively transcribed genes in the liver-lineage cells. These findings provided the evidence suggesting that accumulation of genetic alterations in fetal hepatic progenitor cells progressed to liver cancers, and the selection of mutagenesis depends on active transcription in the liver-lineage cells.


Assuntos
Citidina Desaminase/fisiologia , Neoplasias Hepáticas Experimentais/etiologia , Fígado/citologia , Células-Tronco/fisiologia , Animais , Transformação Celular Neoplásica , Citidina Desaminase/genética , Toxina Diftérica/farmacologia , Camundongos , Camundongos Transgênicos
6.
Gastroenterology ; 146(1): 222-32.e35, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24055508

RESUMO

BACKGROUND & AIMS: Hepatocellular carcinoma develops in patients with chronic hepatitis or cirrhosis via a stepwise accumulation of various genetic alterations. To explore the genetic basis of development of hepatocellular carcinoma in hepatitis C virus (HCV)-associated chronic liver disease, we evaluated genetic variants that accumulate in nontumor cirrhotic liver. METHODS: We determined the whole exome sequences of 7 tumors and background cirrhotic liver tissues from 4 patients with HCV infection. We then performed additional sequencing of selected exomes of mutated genes, identified by whole exome sequencing, and of representative tumor-related genes on samples from 22 cirrhotic livers with HCV infection. We performed in vitro and in vivo functional studies for one of the mutated genes. RESULTS: Whole exome sequencing showed that somatic mutations accumulated in various genes in HCV-infected cirrhotic liver tissues. Among the identified genes, the leptin receptor gene (LEPR) was one of the most frequently mutated in tumor and nontumor cirrhotic liver tissue. Selected exome sequencing analyses detected LEPR mutations in 12 of 22 (54.5%) nontumorous cirrhotic livers. In vitro, 4 of 7 (57.1%) LEPR mutations found in cirrhotic livers reduced phosphorylation of STAT3 to inactivate LEPR-mediated signaling. Moreover, 40% of Lepr-deficient (C57BL/KsJ-db/db) mice developed liver tumors after administration of thioacetamide compared with none of the control mice. CONCLUSIONS: Based on analysis of liver tissue samples from patients, somatic mutations accumulate in LEPR in cirrhotic liver with chronic HCV infection. These mutations could disrupt LEPR signaling and increase susceptibility to hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/genética , Hepatite C Crônica/genética , Cirrose Hepática/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas/genética , Receptores para Leptina/genética , Adulto , Idoso , Animais , Carcinoma Hepatocelular/complicações , Feminino , Predisposição Genética para Doença , Técnicas de Genotipagem , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/complicações , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação
7.
J Clin Microbiol ; 51(11): 3645-52, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23985907

RESUMO

Hepatitis C virus (HCV) reinfects liver allografts in transplant recipients by replicating immediately after transplantation, causing a rapid increase in blood serum HCV RNA levels. We evaluated dynamic changes in the viral genetic complexity after HCV reinfection of the graft liver; we also identified the characteristics of replicating HCV clones using a massively parallel ultradeep sequencing technique to determine the full-genome HCV sequences in the liver and serum specimens of five transplant recipients with genotype 1b HCV infection before and after liver transplantation. The recipients showed extremely high genetic heterogeneity before transplantation, and the HCV population makeup was not significantly different between the liver and blood serum specimens of the individuals. Viral quasispecies complexity in serum was significantly lower after liver transplantation than before it, suggesting that certain HCV clones selectively proliferated after transplantation. Defective HCV clones lacking the structural region of the HCV genome did not increase in number, and full-genome HCV clones selectively increased in number immediately after liver transplantation. A re-increase in the same defective clone existing before transplantation was detected 22 months after transplantation in one patient. Ultradeep sequencing technology revealed that the genetic heterogeneity of HCV was reduced after liver transplantation. Dynamic changes in defective HCV clones after liver transplantation indicate that these clones have important roles in the HCV life cycle.


Assuntos
Variação Genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/genética , Hepatite C Crônica/virologia , Transplante de Fígado , Fígado/virologia , Transplante , Idoso , Sangue/virologia , Feminino , Genótipo , Hepacivirus/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética
8.
Cancer Sci ; 104(10): 1372-9, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23837649

RESUMO

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is an emerging application for lipid research that provides a comprehensive and detailed spatial distribution of ionized molecules. Recent lipidomic approach has identified several phospholipids and phosphatidylinositols (PIs) are accumulated in breast cancer tissues and are therefore novel biomarker candidates. Because their distribution and significance remain unclear, we investigated the precise spatial distribution of PIs in human breast cancer tissues using high-resolution MALDI IMS. We evaluated tissues from nine human breast cancers and one normal mammary gland by negative ion MALDI IMS at a resolution of 10 µm. We detected 10 PIs with different fatty acid compositions, and their proportions were remarkably variable in the malignant epithelial regions. High-resolution imaging enabled us to discriminate cancer cell clusters from the adjacent stromal tissue within epithelial regions; moreover, this technique revealed that several PIs were specifically localized to cancer cell clusters. These PIs were heterogeneously distributed within cancer cell clusters, allowing us to identify two different populations of cancer cells that predominantly expressed either PI(18:0/18:1) or PI(18:0/20:3). Tracing the expression level of PIs during cancer cell progression suggested that the latter population is associated with the invasion. Our study documents a novel model for phospholipid analysis of breast cancer tissues by using high-resolution MALDI IMS and identifies candidate PIs that can describe a specific phenotype of cancer cells.


Assuntos
Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Fosfatidilinositóis/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/química , Neoplasias da Mama/ultraestrutura , Carcinoma Ductal de Mama/ultraestrutura , Progressão da Doença , Células Epiteliais/química , Ácidos Graxos/análise , Feminino , Doença da Mama Fibrocística/metabolismo , Doença da Mama Fibrocística/patologia , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Fenótipo , Pós-Menopausa , Pré-Menopausa , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Células Estromais/química
9.
Anal Bioanal Chem ; 405(12): 4289-93, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23380952

RESUMO

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.


Assuntos
Glicoproteínas/química , Polissacarídeos/análise , Receptor ErbB-2/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Aminoquinolinas/química , Sequência de Carboidratos , Linhagem Celular , Ácidos Cumáricos/química , Glicopeptídeos/química , Glicosilação , Humanos , Dados de Sequência Molecular
10.
Anal Chem ; 84(16): 7146-51, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22830976

RESUMO

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.


Assuntos
Aminoquinolinas/química , Ácidos Cumáricos/química , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Linhagem Celular Tumoral , Glicosilação , Humanos , Polissacarídeos/metabolismo , Receptor ErbB-2/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem , Fatores de Tempo
11.
Proteomics ; 12(12): 2024-35, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22623366

RESUMO

S-Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O(6) -alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR(-/-) ) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR(-/-) mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR(-/-) mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR(-/-) mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR(-/-) mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.


Assuntos
Glutationa Redutase/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/metabolismo , Proteoma/metabolismo , Álcool Desidrogenase , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Estresse do Retículo Endoplasmático/genética , Feminino , Glutationa Redutase/genética , Fígado/química , Fígado/efeitos dos fármacos , Fígado/enzimologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteoma/química , Proteoma/genética , Proteômica
12.
PLoS One ; 7(4): e35052, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22523569

RESUMO

BACKGROUND AND AIMS: Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. METHODS: To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. RESULTS: Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. CONCLUSION: Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.


Assuntos
Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Adulto , Idoso , Antivirais/uso terapêutico , DNA Viral/análise , Feminino , Genoma Viral/genética , Guanina/análogos & derivados , Guanina/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lamivudina/uso terapêutico , Fígado/química , Masculino , Pessoa de Meia-Idade
13.
PLoS One ; 7(2): e31422, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22384020

RESUMO

OBJECTIVE: Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. METHODS AND RESULTS: RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or down-regulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p = 0.005) of the gene expression by interacting with two binding sites in the 3'-UTR of CCNE2. CONCLUSION: In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Regiões 3' não Traduzidas , Antineoplásicos/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Genes Reporter , Humanos , Receptor ErbB-2/biossíntese , Trastuzumab
14.
Proteome Sci ; 10(1): 74, 2012 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-23273257

RESUMO

BACKGROUND: Nitric oxide (NO) mediates its function through the direct modification of various cellular targets. S-nitrosylation is a post-translational modification of cysteine residues by NO that regulates protein function. Recently, an imbalance of S-nitrosylation has also been linked to neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. RESULTS: In the present study, we used two-dimensional gel electrophoresis in conjunction with the modified biotin switch assay for protein S-nitrosothiols using resin-assisted capture (SNO-RAC) to identify proteins that are S-nitrosylated more intensively in neuroblastoma cells treated with a mitochondrial complex I inhibitor, 1-methyl-4-phenylpyridinium (MPP+). We identified 14 proteins for which S-nitrosylation was upregulated and seven proteins for which it was downregulated in MPP+-treated neuroblastoma cells. Immunoblot analysis following SNO-RAC confirmed a large increase in the S-nitrosylation of esterase D (ESD), serine-threonine kinase receptor-associated protein (STRAP) and T-complex protein 1 subunit γ (TCP-1 γ) in MPP+-treated neuroblastoma cells, whereas S-nitrosylation of thioredoxin domain-containing protein 5 precursor (ERp46) was decreased. CONCLUSIONS: These results suggest that S-nitrosylation resulting from mitochondrial dysfunction can compromise neuronal survival through altering multiple signal transduction pathways and might be a potential therapeutic target for neurodegenerative diseases.

15.
PLoS One ; 6(9): e24907, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21966381

RESUMO

BACKGROUND AND AIMS: The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. METHODS: Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy. RESULTS: Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents. CONCLUSION: Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.


Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Idoso , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , Quimioterapia Combinada , Feminino , Heterogeneidade Genética , Genoma Viral/genética , Hepacivirus/genética , Hepatite C/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Taxa de Mutação , Polietilenoglicóis/uso terapêutico , RNA Viral/genética , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Resultado do Tratamento
16.
J Nucleic Acids ; 2011: 131579, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21776374

RESUMO

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

17.
FEBS J ; 278(10): 1598-609, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21395977

RESUMO

MicroRNAs (miRNAs) comprise species of short noncoding RNA that regulate gene expression post-transcriptionally. Recent studies have demonstrated that epigenetic mechanisms, including DNA methylation and histone modification, not only regulate the expression of protein-encoding genes, but also miRNAs, such as let-7a, miR-9, miR-34a, miR-124, miR-137, miR-148 and miR-203. Conversely, another subset of miRNAs controls the expression of important epigenetic regulators, including DNA methyltransferases, histone deacetylases and polycomb group genes. This complicated network of feedback between miRNAs and epigenetic pathways appears to form an epigenetics-miRNA regulatory circuit, and to organize the whole gene expression profile. When this regulatory circuit is disrupted, normal physiological functions are interfered with, contributing to various disease processes. The present minireview details recent discoveries involving the epigenetics-miRNA regulatory circuit, suggesting possible biological insights into gene-regulatory mechanisms that may underlie a variety of diseases.


Assuntos
Epigênese Genética , MicroRNAs/genética , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Regulação da Expressão Gênica , Impressão Genômica , Histona Desacetilases/metabolismo , Humanos , MicroRNAs/metabolismo , Proteínas do Grupo Polycomb , Proteínas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Ribonuclease III/metabolismo
18.
PLoS One ; 6(1): e16435, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21298008

RESUMO

OBJECTIVE: Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection. METHODS: We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC). RESULTS: The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the 'field effect'. CONCLUSION: MicroRNA profiling can predict HCC recurrence in Milan criteria cases.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Perfilação da Expressão Gênica , Neoplasias Hepáticas/diagnóstico , MicroRNAs/análise , Valor Preditivo dos Testes , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/cirurgia , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Prognóstico , Curva ROC , Recidiva , Análise de Sobrevida , Fatores de Tempo
19.
J Hum Genet ; 56(4): 270-6, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21289630

RESUMO

MicroRNAs (miRNAs) are endogenous non-coding RNAs that function as negative regulators of gene expression. Alterations in miRNA expression have been shown to affect tumor growth and response to chemotherapy. In this study, we explored the possible role of miRNAs in cisplatin resistance in esophageal squamous cell carcinoma (ESCC). First we assessed the sensitivity of nine human ESCC cell lines (KYSE series) to cisplatin using an in vitro cell viability assay, and then we compared the miRNA profiles of the cisplatin-sensitive and -resistant cell lines by miRNA microarray analysis. The two groups showed markedly different miRNA expression profiles, and 10 miRNAs were found to be regulated differentially between the two groups. When miR-141, which was the most highly expressed miRNA in the cisplatin-resistant cell lines, was expressed ectopically in the cisplatin-sensitive cell lines, cell viability after cisplatin treatment was increased significantly. Furthermore, we found that miR-141 directly targeted the 3'-untranslated region of YAP1, which is known to have a crucial role in apoptosis induced by DNA-damaging agents, and thus downregulated YAP1 expression. Our study highlights an important regulatory role for miR-141 in the development of cisplatin resistance in ESCC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma de Células Escamosas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Fosfoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Cisplatino/metabolismo , Cisplatino/farmacologia , Primers do DNA/genética , Humanos , MicroRNAs/genética , Análise em Microsséries , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Azul Tripano , Proteínas de Sinalização YAP
20.
Int J Cancer ; 129(9): 2134-46, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21170987

RESUMO

Polo-like kinase 1 (PLK1) is overexpressed in various human cancers. However, the biological functions and the post-transcriptional regulations of PLK1 in esophageal cancer (EC) are still unknown. The purposes of our study are to determine whether PLK1 can be a molecular target of EC therapy and to identify a microRNA (miRNA) targeting PLK1. We performed loss-of-function and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation and luciferase reporter assays, using siRNAs against PLK1 and miRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knockdown of PLK1 in EC cells induced G2/M arrest (p < 0.001) in cell cycle assay and reduced cell proliferation (p = 0.019) and tumor formation ability in vivo (p < 0.0001). MiR-593*, identified as a miRNA targeting PLK1 by a database search, was less expressed especially in six EC cell lines than HEEpiC cells. Moreover, miR-593* expression level was inversely correlated with PLK1 mRNA level in 48 clinical tissue specimens of EC (p = 0.006). Introduction of synthetic miR-593* suppressed PLK1 expression by 69-73%, reduced cell proliferation (p = 0.008) and increased cell proportion of G2/M phase (p = 0.01) in HSA/c (an EC cells), whereas a miR-593* inhibitor upregulated PLK1 expression by 11-55%. Additionally, luciferase assay demonstrated that miR-593* interacted two binding sites in the PLK1 3'-UTR and reduced 56.8-71.5% of luciferase activity by degrading luciferase mRNA in HSA/c cells. In conclusion, PLK1 is post-transcriptionally regulated by miR-593* and could be a promising molecular target for EC treatment.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Estabilidade de RNA , RNA Mensageiro/metabolismo , Quinase 1 Polo-Like
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