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1.
JCI Insight ; 2(1): e91419, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28097239

RESUMO

Peroxisome proliferator-activated receptor-δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity for metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/genética , Metástase Neoplásica/genética , Neoplasias/genética , PPAR delta/genética , Indutores da Angiogênese/metabolismo , Animais , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Deleção de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116/citologia , Células HCT116/metabolismo , Humanos , Interleucina-8/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Terapia de Alvo Molecular/métodos , Metástase Neoplásica/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , PPAR delta/metabolismo
2.
Mol Biol Cell ; 27(8): 1300-9, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26912793

RESUMO

Depletion of microtubule (MT) regulators can initiate stable alterations in MT assembly rates that affect chromosome instability and mitotic spindle function, but the manner by which cellular MT assembly rates can stably increase or decrease is not understood. To investigate this phenomenon, we measured the response of microtubule assembly to both rapid and long-term loss of MT regulators MCAK/Kif2C and Kif18A. Depletion of MCAK/Kif2C by siRNA stably decreases MT assembly rates in mitotic spindles, whereas depletion of Kif18A stably increases rates of assembly. Surprisingly, this is not phenocopied by rapid rapamycin-dependent relocalization of MCAK/Kif2C and Kif18A to the plasma membrane. Instead, this treatment yields opposite affects on MT assembly. Rapidly increased MT assembly rates are balanced by a decrease in nucleated microtubules, whereas nucleation appears to be maximal and limiting for decreased MT assembly rates and also for long-term treatments. We measured amplified tubulin synthesis during long-term depletion of MT regulators and hypothesize that this is the basis for different phenotypes arising from long-term versus rapid depletion of MT regulators.


Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Membrana Celular/metabolismo , Células HCT116/citologia , Células HCT116/efeitos dos fármacos , Células HeLa/citologia , Células HeLa/efeitos dos fármacos , Humanos , Cinesinas/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Fuso Acromático/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo
3.
Am J Pathol ; 186(4): 1015-24, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26835537

RESUMO

Neovascularization drives tumor development, and angiogenic factors are important neovascularization initiators. We recently identified the secreted angiogenic factor CNPY2, but its involvement in cancer has not been explored. Herein, we investigate CNPY2's role in human colorectal cancer (CRC) development. Tumor samples were obtained from CRC patients undergoing surgery. Canopy 2 (CNPY2) expression was analyzed in tumor and adjacent normal tissue. Stable lines of human HCT116 cells expressing CNPY2 shRNA or control shRNA were established. To determine CNPY2's effects on tumor xenografts in vivo, human CNPY2 shRNA HCT116 cells and controls were injected into nude mice, separately. Cellular apoptosis, growth, and angiogenesis in the xenografts were evaluated. CNPY2 expression was significantly higher in CRC tissues. CNPY2 knockdown in HCT116 cells inhibited growth and migration and promoted apoptosis. In xenografts, CNPY2 knockdown prevented tumor growth and angiogenesis and promoted apoptosis. Knockdown of CNPY2 in the HCT116 CRC cell line reversibly increased p53 activity. The p53 activation increased cyclin-dependent kinase inhibitor p21 and decreased cyclin-dependent kinase 2, thereby inhibiting tumor cell growth, inducing cell apoptosis, and reducing angiogenesis both in vitro and in vivo. CNPY2 may play a critical role in CRC development by enhancing cell proliferation, migration, and angiogenesis and by inhibiting apoptosis through negative regulation of the p53 pathway. Therefore, CNPY2 may represent a novel CRC therapeutic target and prognostic indicator.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Feminino , Células HCT116/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Prognóstico , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
4.
J Biotechnol ; 205: 24-35, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25592049

RESUMO

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.


Assuntos
Técnicas de Cocultura/métodos , Fígado/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Esferoides Celulares/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Antineoplásicos Alquilantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/instrumentação , Ciclofosfamida/farmacologia , Células HCT116/citologia , Células HCT116/efeitos dos fármacos , Humanos , Técnicas Analíticas Microfluídicas/métodos , Ratos , Esferoides Celulares/efeitos dos fármacos , Técnicas de Cultura de Tecidos/instrumentação
5.
Asian Pac J Cancer Prev ; 15(20): 8685-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25374190

RESUMO

The objective of the present study was to investigate cloning, expression, and functions of the recombinant protein, Siva1. Siva1 gene was synthesized by RT-PCR from HCT116 cells. Plasmids were cleaved with the restriction endonuclease, BamH1/Sal1 and products were connected to pQE30, which underwent cleavage by BamH1/Sal1. The recombinant plasmid, pQE30-Siva1, was identified after digestion with restriction endonucleases followed by transformation into E. coli M15. Expression of Siva1 was induced by IPTG and identified by SDS- PAGE following purification with affinity chromatography. The results showed that size of Siva1 was 12 kDa, consistent with the molecular weight of the His-Siva1 fusion protein. Functional test demonstrated that Siva1 significantly inhibited the invasion and migration of HCT116 cells. It may thus find clinical application for control of cancers.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/isolamento & purificação , Clonagem Molecular/métodos , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Engenharia Genética/métodos , Células HCT116/citologia , Células HCT116/fisiologia , Humanos , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes de Fusão/genética , Sensibilidade e Especificidade , Células Tumorais Cultivadas
6.
Genes Cells ; 18(11): 1042-52, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24165024

RESUMO

Cell-in-cell structures represent live cell events in which one cell internalizes another. Because formation of cell-in-cell structures is a rare event in most cell types and the event is associated with cell death, there has been limited clarification of this phenomenon, and its physiological role and molecular mechanism are yet to be precisely elucidated. In this study, we established a mutagenized cell line that exhibited cell-in-cell structures at a more than 10-fold higher frequency as compared to the parent cells. Interestingly, both engulfment and invasion were increased in the mutagenized cell line as compared with that in the parent cell line in the suspension culture condition. This finding indicates that this mutagenized cell line showed an interchangeable status in terms of its ability to form cell-in-cell structures, and the system described here could be useful for elucidation of the mechanisms regulating the formation of cell-in-cell structures, including engulfment and invasion, in a given cellular environment. Further studies using this cell line are warranted to understand the mechanism of formation and biological significance of the cell-in-cell formation.


Assuntos
Formação de Célula em Célula , Células HCT116/citologia , Aminacrina/análogos & derivados , Aminacrina/farmacologia , Células HCT116/metabolismo , Humanos , Mutagênese , Taxa de Mutação , Compostos de Mostarda Nitrogenada/farmacologia , Fenótipo
7.
Cancer Biol Ther ; 7(12): 2047-53, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19106634

RESUMO

Activation of the PI3K/Akt pathway is associated with tumorigenesis and resistance to apoptosis and ionizing radiation (IR). We sought to characterize the effects of physiologic and genetic manipulation of Akt signaling on IR-induced gastrointestinal (GI) apoptosis in mice. PI3K/Akt signaling is stimulated by insulin. We evaluated the time course of Akt stimulation by insulin and found it overlapped with protection from apoptosis induced by TRAIL (TNFalpha Related Apoptosis Inducing Ligand) in cell lines. Mice were treated with insulin and glucose and the kinetics of in vivo Akt stimulation were determined by phospho-Akt (S473) (P-Akt) immunofluorescence in the gut. Irradiation of mice by five Gy at 30 minutes after insulin/glucose administration induced apoptosis in the crypts of the ileum and colon after six hours, but induced little apoptosis in the liver or esophagus. Pre-treatment with insulin and glucose did not significantly alter levels of IR-induced apoptosis in the gut. IR alone led to sustained increases in P-Akt in the gut at six hours, a protective response that may have precluded additional protection from insulin/glucose. In Akt1-/- mice, there was significantly more apoptosis in ileum crypts of irradiated mice compared to Akt1+/+ mice, suggesting a role for the pathway in the GI tract in response to IR. Taken together, modulation of the PI3K/Akt pathway may sensitize or protect against cancer therapies in both tumor and normal tissues.


Assuntos
Apoptose/fisiologia , Neoplasias Gastrointestinais/patologia , Células HCT116/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Células HCT116/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/efeitos da radiação , Transdução de Sinais
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