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1.
Oncotarget ; 8(13): 21315-21326, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28423509

RESUMO

Histone H3 lysine 9 dimethylation (H3K9me2) is mainly regulated by the histone lysine methyltransferase G9a and is associated with the repression of transcription. However, both the role of G9a and the significance of H3K9me2 in hepatocellular carcinoma (HCC) cells remain unclear. In this study, we conducted loss-of-function assay of G9a using short-hairpin RNA and pharmacological interference. Knockdown of G9a reduced H3K9me2 levels and impaired both HCC cell growth and sphere formation. However, transforming growth factor ß1-induced epithelial mesenchymal transition (EMT) was not suppressed by G9a knockdown. Combined analyses of chromatin immunoprecipitation followed by sequencing and RNA-sequencing led to successful identification of 96 candidate epigenetic targets of G9a. Pharmacological inhibition of G9a by BIX-01294 resulted in both cell growth inhibition and induction of apoptosis in HCC cells. Intraperitoneal administration of BIX-01294 suppressed the growth of xenograft tumors generated by implantation of HCC cells in non-obese diabetic/severe combined immunodeficient mice. Immunohistochemical analyses revealed high levels of G9a and H3K9me2 in 36 (66.7%) and 35 (64.8%) primary HCC tissues, respectively. G9a expression levels were significantly positively correlated with H3K9me2 levels in tumor tissues. In contrast, in non-tumor tissues, G9a and H3K9me2 were only observed in biliary epithelial cells and periportal hepatocytes. In conclusion, G9a inhibition impairs anchorage-dependent and -independent cell growth, but not EMT in HCC cells. Our data indicate that pharmacological interference of G9a might be a novel epigenetic approach for the treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Neoplasias Hepáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Azepinas/farmacologia , Western Blotting , Carcinoma Hepatocelular/patologia , Imunoprecipitação da Cromatina , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade , Histonas/genética , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Quinazolinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Int J Cancer ; 136(2): 289-98, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24844570

RESUMO

Histone H3 lysine 9 trimethylation (H3K9me3) is associated with transcriptional repression and regulated by histone lysine methyltransferases such as SUV39H1 and ESET. However, the functional roles of these enzymes in hepatocellular carcinoma (HCC) remain uncertain. In this study, we conducted loss-of-function assays for HCC cells. SUV39H1 knockdown but not ESET knockdown reduced H3K9me3 levels and impaired HCC cell growth and sphere formation. The pharmacological inhibition of SUV39H1 by chaetocin resulted in cell growth inhibition and inducing cellular apoptosis in culture and xenograft subcutaneous tumors. Real-time polymerase chain reaction analysis indicated high levels of SUV39H1 expression in 24 of 42 (57.1%) HCC surgical samples compared with corresponding nontumor tissues. Immunohistochemistry identified high levels of H3K9me3 and ESET proteins in 23 (54.8%) and 29 (69.0%) tumor tissues, respectively. However, these proteins' expressions were only observed in biliary epithelial cells and periportal hepatocytes of nontumor tissues. Expression levels of SUV39H1 but not those of ESET were significantly correlated with H3K9me3 levels. The cumulative HCC recurrence rate was significantly higher for patients with elevated SUV39H1 expression and H3K9me3 levels. In conclusion, our data indicate that elevated SUV39H1 expression and high levels of H3K9me3 have important roles in HCC development and progression. Therefore, the pharmacological inhibition of SUV39H1 may be a promising therapeutic approach for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Epigênese Genética , Histonas/metabolismo , Neoplasias Hepáticas/genética , Metiltransferases/genética , Recidiva Local de Neoplasia/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Western Blotting , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Metilação de DNA , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS One ; 9(1): e84807, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24454751

RESUMO

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)(+) HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM(+) cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM(+) cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.


Assuntos
Carcinoma Hepatocelular/enzimologia , Dissulfiram/farmacologia , Neoplasias Hepáticas/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dissulfiram/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glipicanas/genética , Glipicanas/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS One ; 8(7): e70010, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23922888

RESUMO

Metformin has been widely used as an oral drug for diabetes mellitus for approximately 60 years. Interestingly, recent reports showed that metformin exhibited an anti-tumor action in a wide range of malignancies including hepatocellular carcinoma (HCC). In the present study, we investigated its impact on tumor-initiating HCC cells. Metformin suppressed cell growth and induced apoptosis in a dose-dependent manner. Flow cytometric analysis showed that metformin treatment markedly reduced the number of tumor-initiating epithelial cell adhesion molecule (EpCAM)(+) HCC cells. Non-adherent sphere formation assays of EpCAM(+) cells showed that metformin impaired not only their sphere-forming ability, but also their self-renewal capability. Consistent with this, immunostaining of spheres revealed that metformin significantly decreased the number of component cells positive for hepatic stem cell markers such as EpCAM and α-fetoprotein. In a xenograft transplantation model using non-obese diabetic/severe combined immunodeficient mice, metformin and/or sorafenib treatment suppressed the growth of tumors derived from transplanted HCC cells. Notably, the administration of metformin but not sorafenib decreased the number of EpCAM(+) cells and impaired their self-renewal capability. As reported, metformin activated AMP-activated protein kinase (AMPK) through phosphorylation; however its inhibitory effect on the mammalian target of rapamycin (mTOR) pathway did not necessarily correlate with its anti-tumor activity toward EpCAM(+) tumor-initiating HCC cells. These results indicate that metformin is a promising therapeutic agent for the elimination of tumor-initiating HCC cells and suggest as-yet-unknown functions other than its inhibitory effect on the AMPK/mTOR pathway.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Metformina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
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