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1.
BMC Cancer ; 9: 240, 2009 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-19615090

RESUMO

BACKGROUND: Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC). METHODS: We examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion. RESULTS: We detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively. CONCLUSION: Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular/fisiologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/biossíntese , Proteína 1 Relacionada a Twist/biossíntese , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Nucleares/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteína 1 Relacionada a Twist/genética
2.
Mol Oncol ; 2(3): 241-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19383345

RESUMO

Using GGCX gene-specific real-time PCR, exon 2 deletion splice variant of vitamin K-dependent gamma-glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK-Hep-1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP-producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP-negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines.


Assuntos
Carbono-Carbono Ligases/fisiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Precursores de Proteínas/biossíntese , Protrombina/biossíntese , Biomarcadores , Carbono-Carbono Ligases/genética , Carcinoma Hepatocelular/enzimologia , Linhagem Celular Tumoral , Éxons , Variação Genética , Humanos , Isoenzimas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise
3.
J Biol Chem ; 282(12): 8741-8, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17255102

RESUMO

Des-gamma-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma. Previously, we have demonstrated that DCP stimulates cell proliferation in hepatocellular carcinoma cell lines through Met-Janus kinase 1 signal transducer and activator of transcription 3 signaling pathway. In the present study, we demonstrated that DCP induces both cell proliferation and migration in human umbilical vein endothelial cells. DCP was found to bind with the kinase insert domain receptor (KDR), alternatively referred to as vascular endothelial growth factor receptor-2. Furthermore, DCP induced autophosphorylation of KDR and its downstream effector phospholipase C-gamma and mitogen-activated protein kinase (MAPK). To support these results, we showed that DCP-induced cell proliferation and cell migration were inhibited by KDR short interfering RNA, KDR kinase inhibitor, or MAPK inhibitor. In conclusion, these results indicate that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities.


Assuntos
Biomarcadores/química , Endotélio Vascular/citologia , Precursores de Proteínas/química , Protrombina/química , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Inativação Gênica , Humanos , Janus Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfolipase C gama/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Veias Umbilicais/citologia
5.
Neurobiol Dis ; 18(1): 143-51, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15649705

RESUMO

Parkinson's disease is characterized by selective loss of dopaminergic neurons in the substantia nigra. However, its associated cell death mechanism remains unknown. 1-Methyl-4-phenil-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) cause dopaminergic neuronal cell death. Both are widely used to model PD. We investigated the role of a vesicle-transport-related protein, RA410/Sly1, in SH-SY5Y cells to clarify the mechanism of cellular adaptation to MPP+ and 6-OHDA-induced stress. Antisense RA410/Sly1 transformants treated with these toxins displayed reduced viability in comparison with viability of wild-type or RA410/Sly1 sense transformants. Electron microscopy analysis indicated that the ER in MPP+-treated antisense RA410/Sly1 transformants was rapidly disrupted in comparison to wild-type or sense RNA transformants. Cell death induced by MPP+ and 6-OHDA was suppressed in RA410/Sly1 sense transformants through suppression of caspase-2, -3 and -9 activation. These results suggest that RA410/Sly1 plays an important cytoprotective role in MPP+ and 6-OHDA-induced cellular perturbation.


Assuntos
Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Degeneração Neural/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/fisiologia , 1-Metil-4-fenilpiridínio/toxicidade , Adaptação Fisiológica/fisiologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dopamina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Microscopia Eletrônica de Transmissão , Proteínas Munc18 , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/ultraestrutura , Oligonucleotídeos Antissenso/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/toxicidade , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Ratos , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/fisiopatologia , Transfecção , Células Tumorais Cultivadas
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