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1.
Int J Oncol ; 61(3)2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35894141

RESUMO

Pyruvate dehydrogenase kinase 4 (PDK4) is an important regulator of energy metabolism. Previously, knockdown of PDK4 by specific small interfering RNAs (siRNAs) have been shown to suppress the expression of Κirsten rat sarcoma viral oncogene homolog (KRAS) and the growth of lung and colorectal cancer cells, indicating that PDK4 is an attractive target of cancer therapy by altering energy metabolism. The authors previously reported that a novel small molecule, cryptotanshinone (CPT), which inhibits PDK4 activity, suppresses the in vitro three­dimensional (3D)­spheroid formation and in vivo tumorigenesis of KRAS­activated human pancreatic and colorectal cancer cells. The present study investigated the molecular mechanism of CPT­induced tumor suppression via alteration of glutamine and lipid metabolism in human pancreatic and colon cancer cell lines with mutant and wild­type KRAS. The antitumor effect of CPT was more pronounced in the cancer cells containing mutant KRAS compared with those containing wild­type KRAS. CPT treatment decreased glutamine and lipid metabolism, affected redox regulation and increased reactive oxygen species (ROS) production in the pancreatic cancer cell line MIAPaCa­2 containing mutant KRAS. Suppression of activated KRAS by specific siRNAs decreased 3D­spheroid formation, the expression of acetyl­CoA carboxylase 1 and fatty acid synthase (FASN) and lipid synthesis. The suppression also reduced glutathione­SH/glutathione disulfide and increased the production of ROS. Knockdown of FASN suppressed lipid synthesis in MIAPaCa­2 cells, partially promoted ROS production and mildly suppressed 3D­spheroid formation. These results indicated that CPT reduced tumorigenesis by inhibiting lipid metabolism and promoting ROS production in a mutant KRAS­dependent manner. This PDK4 inhibitor could serve as a novel therapeutic drug for KRAS­driven intractable cancers via alteration of cell metabolism.


Assuntos
Neoplasias Colorretais , Neoplasias Pancreáticas , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/patologia , Glutamina/metabolismo , Humanos , Lipídeos , Lipogênese , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenantrenos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Neoplasias Pancreáticas
2.
Int J Oncol ; 59(1)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33982789

RESUMO

The phosphorylation of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase (PDK) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is upregulated in various types of human cancer. Because PDK4 regulation is critical for metabolic changes in cancer cells, it is an attractive target for cancer therapy given its ability to shift glucose metabolism. It was previously shown that a novel PDK4 inhibitor, cryptotanshinone (CPT), suppressed the three­dimensional (3D)­spheroid formation of pancreatic and colorectal cancer cells. In the present study, the effects of CPT on the invasiveness of bladder cancer cells were investigated. CPT significantly suppressed the invasiveness and 3D­spheroid formation of T24 and J82 bladder cancer cells. CPT also suppressed the phosphorylation of PDH and ß­catenin, as well as the expression of N­cadherin, which are all critical for inducing epithelial­mesenchymal transition (EMT). The knockdown of ß­catenin or PDK4 using specific small interfering RNAs suppressed N­cadherin expression and invasiveness in T24 cells. An mTOR inhibitor also suppressed the phosphorylation of ß­catenin and N­cadherin expression. Furthermore, CPT injection significantly suppressed pancreatic tumor growth and peritoneal dissemination of highly metastatic SUIT­2 pancreatic cancer cells in a mouse orthotopic pancreatic cancer model, without evident toxicity. Moreover, immunohistochemistry analyses demonstrated decreased ß­catenin expression in CPT­treated pancreatic tumors compared with control tumors. Taken together, these results indicate that CPT reduced the invasiveness and metastasis of bladder cancer cells by suppressing EMT via the mTOR/ß­catenin/N­cadherin pathway.


Assuntos
Antineoplásicos/farmacologia , Fenantrenos/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
3.
Mol Carcinog ; 58(10): 1726-1737, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31106493

RESUMO

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.


Assuntos
Família Aldeído Desidrogenase 1/genética , Inibidores Enzimáticos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Retinal Desidrogenase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/genética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Int J Oncol ; 52(1): 231-240, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115414

RESUMO

The human cyclin D1 gene generates two major isoforms, cyclin D1a and cyclin D1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclin D1b siRNA, we investigated the anti-oncogenic effects of cyclin D1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclin D1b mRNA. Knockdown of cyclin D1b by specific siRNA significantly suppressed cell proliferation, in vitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclin D1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclin D1b siRNA showed that cyclin D1b siRNA induced apoptosis. In T24 cells, knockdown of cyclin D1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclin D1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclin D1b siRNA. Taken together, these results indicate that knockdown of cyclin D1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclin D1b siRNA will be a novel therapy for cyclin D1b-expressing bladder cancers.


Assuntos
Ciclina D1/genética , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi/métodos , Neoplasias da Bexiga Urinária/terapia , Animais , Apoptose/genética , Testes de Carcinogenicidade , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Distribuição Aleatória , Transfecção , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Oncol Lett ; 13(6): 4276-4284, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28599427

RESUMO

Periostin is an extracellular matrix protein involved in the regulation of intercellular adhesion. The present study investigated the in vivo tumor suppressor function of periostin in a mouse orthotopic model of bladder cancer. Retroviral vectors were used to transfect human bladder cancer UMUC-3 cell line with periostin. Bladders of nude mice that were transurethrally instilled with periostin-expressing UMUC-3 cells were revealed to weigh less compared with bladders instilled with vector control cells. In total, five (83.3%) of six vector control UMUC-3 bladder tumors exhibited histological evidence of muscle invasion. However, none of the five periostin-expressing UMUC-3 bladder tumors revealed muscle invasion. Thick edematous lesions were present in the submucosa of periostin-expressing UMUC-3 bladder tumors. The expression of periostin also suppressed in vitro cell invasiveness of UMUC-3 cells without affecting cellular proliferation. The level of phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), protein kinase B (Akt) and S6 ribosomal protein, a downstream protein of mammalian target of rapamycin (mTOR) was decreased in periostin-expressing UMUC-3 cells compared with vector control cells. Treatment with 100 ng/ml recombinant human periostin protein also suppressed cell invasiveness and phosphorylation of PDK1, Akt and S6 in UMUC-3 cells, consistent with results using periostin-expressing UMUC-3 cells. Treatment with PDK1, Akt and mTOR inhibitors significantly suppressed UMUC-3 cell invasiveness. These results demonstrate that periostin suppresses in vivo and in vitro invasiveness of bladder cancer via the PDK1/Akt/mTOR signaling pathway. Periostin may be useful as a potent chemotherapeutic agent by suppressing bladder cancer invasiveness.

6.
Mol Carcinog ; 55(1): 52-63, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25620379

RESUMO

Previously, we showed that drs contributes to suppression of malignant tumor formation in drs-knockout (KO) mice. In this study, we demonstrate the regulation of glucose metabolism by drs using comparisons of drs-KO and wild-type (WT) mouse embryonic fibroblasts (MEFs). Extracellular acidification, lactate concentration, and glucose consumption in drs-KO cells were significantly greater than those in WT cells. Metabolomic analyses also confirmed enhanced glycolysis in drs-KO cells. Among glycolysis-regulating proteins, expression of lactate dehydrogenase (LDH)-B was upregulated at the post-transcriptional level in drs-KO cells and increased LDH-B expression, LDH activity, and acidification of culture medium in drs-KO cells were suppressed by retroviral rescue of drs, indicating that LDH-B plays a critical role for glycolysis regulation mediated by drs. In WT cells transformed by activated K-ras, expression of endogenous drs mRNA was markedly suppressed and LDH-B expression was increased. In human cancer cell lines with low drs expression, LDH-B expression was increased. Database analyses also showed the correlation between downregulation of drs and upregulation of LDH-B in human colorectal cancer and lung adenocarcinoma tissues. Furthermore, an LDH inhibitor suppressed anchorage-independent growth of human cancer cells and MEF cells transformed by activated K-ras. These results indicate that drs regulates glucose metabolism via LDH-B. Downregulating drs may contribute to the Warburg effect, which is closely associated with malignant progression of cancer cells.


Assuntos
Genes Supressores de Tumor , Glucose/metabolismo , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Feminino , Técnicas de Inativação de Genes , Humanos , Isoenzimas/metabolismo , Masculino , Metaboloma , Metabolômica/métodos , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo
7.
Oncol Lett ; 12(6): 4850-4856, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28105192

RESUMO

A total of two major isoforms, cyclin D1a and cyclin D1b, are generated from the human cyclin D1 gene by alternative splicing. Cyclin D1b is scarcely expressed in normal tissues; however, it is expressed at a high frequency in certain types of cancerous tissue. The present authors previously constructed cyclin D1b transgenic (Tg) mice and identified rectal tumors, including adenocarcinoma and sessile serrated adenoma, in 62.5% of female Tg mice. In addition, the present authors indicated that cyclin D1b expression enhances phosphorylation of extracellular signal-regulated kinase (Erk) in these rectal tumors, and in mouse embryonic fibroblast (MEF) cells and human 293T cells. In the present study, it was initially demonstrated that cyclin D1b has the ability to enhance cell invasiveness by itself; it additionally increases cell invasiveness, anchorage-independent growth and tumorigenicity in cooperation with an activated K-ras oncogene in MEF cells. Phosphorylation of Akt was increased in cyclin D1b-expressing MEF cells and in the rectal tumor tissues of cyclin D1b Tg mice. Phosphorylation of Akt was also enhanced by transfection of cyclin D1b, but not cyclin D1a, in human 293T cells. Treatment with an Akt inhibitor suppressed phosphorylation of Erk in 293T cells expressing cyclin D1b and D1bTgRT cells established from rectal cancer of the cyclin D1b Tg mouse. Furthermore, the Akt inhibitor suppressed the invasiveness of D1bTgRT cells and the tumor growth of these cells in nude mice when the Akt inhibitor was injected into the tumors. These results indicate that cyclin D1b activates Erk through Akt, and that activation of Akt contributes to the tumorigenicity of the cyclin D1b Tg mice. Inhibitors targeting the phosphoinositide 3-kinase/Akt signaling pathway are thus expected to have therapeutic potential in a variety of human cancer types expressing cyclin D1b.

8.
Carcinogenesis ; 35(1): 227-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23975835

RESUMO

Human cyclin D1 generates two major isoforms via alternative splicing: cyclin D1a and cyclin D1b. Cyclin D1b is hardly expressed in normal tissues but is frequently expressed in certain types of cancer tissues. To clarify the oncogenic potential of cyclin D1b variant, we developed cyclin D1b transgenic (Tg) mice and analyzed their phenotypes. We detected rectal tumors in 63% (15/24) of the female Tg mice. All rectal tumors had the histological characteristics similar to human sessile serrated adenoma/polyps (SSA/Ps). Adenocarcinomas were also found in 53% (8/15) of the rectal tumors, suggesting that these adenocarcinomas originated from the SSA/P-like lesions. No rectal tumors were found in the ovariectomized female cyclin D1b Tg mice (0/10), indicating that ovarian hormones played a critical role in rectal carcinogenesis in these Tg mice. Both phosphorylation of Erk, without activating MEK, and expression of estrogen receptor ß were elevated in the rectal tumors of female cyclin D1b Tg mice compared with normal rectums of female wild-type mice. In addition, we established a cell line, D1bTgRT, derived from a rectal cancer of female Tg mouse. Small interfering RNA-induced cyclin D1b knockdown in this cell line suppressed Erk phosphorylation, anchorage-independent growth, cell invasiveness and tumorigenicity in nude mice. In humans, expression of cyclin D1b messenger RNA was detected in 17% (1/6) of colorectal cancer cell lines and 9.7% (3/31) of colorectal cancer tissues. Taken together, these results indicate that cyclin D1b expression contributes to the female- specific rectal carcinogenesis in mouse model.


Assuntos
Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Retais/patologia , Animais , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Hormônios Esteroides Gonadais/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ovariectomia , Neoplasias Retais/genética , Neoplasias Retais/cirurgia
9.
Cancer Lett ; 314(1): 82-91, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21983129

RESUMO

The drs gene is an apoptosis-inducing tumor suppressor. By using drs-knockout (KO) mouse embryonic fibroblasts (MEFs), we showed that drs is involved in the host defense against viral infection. In drs-KO MEFs infected with vesicular stomatitis virus, the viral replication and protein synthesis were markedly enhanced without the upregulation of the cellular protein synthesis. Phosphorylation of S6K, S6, 4EBP1 and TSC2 proteins was closely correlated with the enhanced viral replication in drs-KO MEFs. Drs protein could associate with stress-inducible GADD34 to form a complex with TSC1/2, which suppresses mTOR activity. These findings indicate that Drs suppresses viral replication via mTOR-dependent pathway.


Assuntos
Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Viroses/imunologia , Replicação Viral , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Proteína Fosfatase 1/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa , Vírus da Estomatite Vesicular Indiana/fisiologia
10.
Int J Oncol ; 38(6): 1759-66, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21468544

RESUMO

We previously showed that periostin expression is downregulated in human bladder cancer tissues and that ectopic expression of periostin suppresses the invasiveness of bladder cancer cells. However, in most other human cancers studied, the expression of periostin promotes cell invasiveness. In the present study, we investigated the regulation of the epithelial-to-mesenchymal transition (EMT) and cell invasiveness by periostin in bladder and prostate cancer cell lines, and found opposite regulation of EMT and cell invasiveness by periostin. Periostin upregulated E-cadherin expression in bladder cancer cells but downregulated it in prostate cancer cells. Periostin suppressed cell invasiveness in bladder cancer cells but promoted it in prostate cancer cells. Snail, a negative regulator of E-cadherin, was upregulated by periostin in prostate cancer cells, while Twist, another negative regulator of E-cadherin, was downregulated in bladder cancer cells. The C-terminal region of periostin was sufficient for these functions in bladder cancer cells but not in prostate cancer cells. Knockdown of endogenous Snail by siRNA suppressed cell invasiveness in prostate cancer cells expressing periostin. Periostin also suppressed Akt phosphorylation in bladder cancer cells but enhanced it in prostate cancer cells. Treatment with Akt inhibitor increased E-cadherin expression and suppressed both Twist expression and cell invasiveness of bladder cancer cells. These results indicate that Akt signaling plays a role in the cell-type-dependent regulation of E-cadherin expression and cell invasiveness by periostin via Snail and Twist.


Assuntos
Moléculas de Adesão Celular/metabolismo , Desdiferenciação Celular , Neoplasias da Próstata/patologia , Neoplasias da Bexiga Urinária/patologia , Animais , Caderinas/genética , Caderinas/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/farmacologia , Desdiferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Camundongos , Células NIH 3T3 , Invasividade Neoplásica/genética , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias da Bexiga Urinária/genética
11.
Mol Carcinog ; 48(10): 953-64, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19415719

RESUMO

Alternative splicing in the cyclin D1 gene produces cyclin D1b variant which lacks a C-terminal region containing the threonine-286 (T286) phosphorylation site required for nuclear export. We have shown that the expression of the cyclin D1b variant is detected in about 60% of human bladder cancer tissues (15/26) and cell lines (3/5). To examine the role of the cyclin D1b variant in bladder carcinogenesis, we introduced wild-type cyclin D1a, cyclin D1b variant or mutant cyclin D1-T286A cDNAs into a human bladder cancer cell line, SBT991, in which cyclin D1b transcript was not expressed, and compared their oncogenic activities. Ectopic expression of cyclin D1b promoted cell invasiveness and anchorage-independent growth of the cancer cells. On the other hand, cyclin D1-T286A enhanced anchorage-independent growth, but did not promote cell invasiveness. The amount of nuclear-localized cyclin D1b and cyclin D1-T286A was higher than that of nuclear-localized cyclin D1a. In addition, introduction of siRNA specific for cyclin D1b into cells of the T24 bladder cancer cell line, in which cyclin D1b transcript was expressed, significantly suppressed cell invasiveness. Immunoprecipitation analysis revealed that cyclin D1a and cyclin D1-T286A could bind to cyclin-dependent kinase 4 (CDK4) but cyclin D1b has lost its capacity to associate with CDK4. Unlike cyclin D1a and cyclin D1-T286A, expression of cyclin D1b did not enhance phosphorylation of Rb protein in SBT991 cells. These results indicate that cyclin D1b promotes cell invasiveness independent of binding to CDK4 to enhance Rb phosphorylation.


Assuntos
Ciclina D1/fisiologia , Quinase 4 Dependente de Ciclina/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Western Blotting , Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosforilação , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Transfecção
12.
Cancer Lett ; 283(1): 74-83, 2009 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-19368996

RESUMO

The drs gene is an apoptosis-inducing tumor suppressor. Previously, we showed that drs contributes to the suppression of tumor formation by generating drs-knockout mice. In this study, by using drs KO mouse embryonic fibroblasts, we found that drs is involved in the autophagy regulation under low serum culture conditions. Both electron microscopy and GFP-LC3 analyses demonstrated that drs is involved in the maturation process of autophagy from autophagosomes to autolysosomes. In addition, drs could associate with Rab24 and the association between drs and Rab24 was enhanced during autophagy. Drs was also co-localized with Rab24 on punctuated structures during autophagy.


Assuntos
Autofagia/fisiologia , Fibroblastos/metabolismo , Genes Supressores de Tumor/fisiologia , Proteínas de Membrana/metabolismo , Fagossomos/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Impressões Digitais de DNA , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fagossomos/genética , Soro , Proteínas rab de Ligação ao GTP/metabolismo
13.
Int J Oncol ; 32(1): 161-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18097555

RESUMO

We have previously reported that the expression of periostin mRNA is significantly repressed in human bladder cancer tissues, and that periostin plays a role as a suppressive factor for invasion and metastasis in the progression of human bladder cancers. In this study, to clarify the role of alternative splicing of periostin in human bladder carcinogenesis, we examined the expression of wild-type (WT) and spliced variants of periostin mRNA in normal bladder and bladder cancer tissues. Although both WT and spliced periostin mRNA were expressed in all normal bladder tissues examined, no WT periostin mRNA was detected in the examined transitional cell carcinomas (TCCs) of the bladder (0/23) or in bladder cancer cell lines (0/6). Spliced variants of periostin were detected in 48% (11/23) of TCC tissues and 33% (2/6) of bladder cancer cell lines. Two types of spliced periostin (Variants I and II) were successfully isolated from bladder cancer tissues, but Variant I, which is predominantly expressed in bladder cancer tissues, did not show suppressor activity on in vitro invasiveness and in vivo metastasis of cancer cells. Immunohistochemical analysis indicated that strong belt-like expression of periostin protein was observed in the stroma just beneath the normal bladder epithelium, while it was mostly attenuated in bladder cancer tissues. These results indicate that the loss of WT periostin by down-regulation and/or alternative splicing, which produces Variant I, is closely correlated with the development of bladder cancer.


Assuntos
Processamento Alternativo , Moléculas de Adesão Celular/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Animais , Moléculas de Adesão Celular/análise , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Invasividade Neoplásica , Bexiga Urinária/metabolismo
14.
J Virol ; 81(20): 11106-15, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17670836

RESUMO

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.


Assuntos
Antígenos de Diferenciação/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico/metabolismo , Replicação Viral , Animais , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/virologia , Imunidade , Camundongos , Fosforilação , Proteína Fosfatase 1 , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR
15.
Int J Oncol ; 30(6): 1343-8, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17487354

RESUMO

Although adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-cell leukemia virus (HTLV-1), many other factors are thought to be required for the progression from indolent ATL to aggressive ATLL. The drs gene was originally isolated as a novel suppressor gene of v-src transformation and was shown to induce apoptosis in human cancer cells. To investigate the involvement of drs downregulation in the progression of ATLL, we examined the expression of drs in smoldering, chronic and aggressive ATLL, and found that drs expression was markedly reduced in clinically aggressive ATLL. In aggressive ATLL cell lines, expression of drs mRNA was not detected, although expression of drs mRNA was detected in T-cell lines infected with HTLV-1. A correlation between drs downregulation and expression of the Tax gene was not observed in these T-cell lines. Furthermore, introduction of drs into an ATL cell line, HUT102, by retrovirus vector suppressed the colony formation of the cells in soft agar and enhanced apoptotic cell death of the cells under low serum culture conditions. These results indicate that downregulation of drs is closely linked to the progression of ATLL, independently of Tax expression, suggesting that drs may suppress the progression of ATLL via enhancing apoptosis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Proteínas de Membrana/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Int J Mol Med ; 19(3): 475-83, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17273797

RESUMO

Cells regulate the rate of protein synthesis during conditions of cell stress to adapt to environmental changes. However, the molecular interactions between signaling pathways controlling translation and the cellular response to stress remain to be elucidated. Here, we show that the expression of growth arrest and DNA damage protein 34 (GADD34) is induced by energy depletion and that the expression of this protein protects cells from apoptotic cell death. During conditions of cell stress, GADD34 forms a stable complex with tuberous sclerosis complex (TSC) 1/2, causes TSC2 dephosphorylation, and inhibits signaling by mammalian target of the rapamycin (mTOR). These findings demonstrate that crosstalk between GADD34 and the mTOR signaling pathways contributes to the response of the protein synthetic machinery to environmental stress. GADD34 may find clinical potential as a target drug for the treatment of mTOR-associated diseases.


Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metabolismo Energético , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Antígenos de Diferenciação/genética , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/deficiência , Glucose/farmacologia , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Ubiquitina/metabolismo
17.
Carcinogenesis ; 28(4): 777-84, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17088259

RESUMO

The drs gene was originally isolated as a suppressor of v-src transformation. Expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and tissues, suggesting the potential role of this gene as a tumor suppressor. Previously, we found that Drs protein associates with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activates caspases to induce apoptosis in human cancer cells without involvement of the mitochondria. In this study, we investigated the tumor suppressor function of drs and the correlation between Drs-mediated apoptosis and tumor suppression by generating a gene-knockout (KO) mouse. Between 7 and 12 months after birth, malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of the drs KO mice, whereas no tumors were found in any of the wild-type mice during the same period of time. drs KO embryonic fibroblasts also showed enhanced sensitivity to transformation by v-src oncogene. Reintroduction of drs into a tumor cell line derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis and the activation of caspase-9 and -3. Furthermore, introduction of drs into this cell line enhanced sensitivity to apoptosis mediated by caspase-3, -9 and -12 under low serum culture conditions. The present results thus indicate that drs contributes to the suppression of malignant tumor formation, and this suppression is closely correlated with drs-mediated apoptosis.


Assuntos
Adenocarcinoma/patologia , Apoptose/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Neoplasias Pulmonares/patologia , Linfoma/patologia , Proteínas de Membrana/genética , Adenocarcinoma/etiologia , Animais , Caspases/metabolismo , Transformação Celular Neoplásica , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Supressores de Tumor , Humanos , Técnicas Imunoenzimáticas , Rim/metabolismo , Neoplasias Pulmonares/etiologia , Linfoma/etiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Transfecção
18.
Int J Cancer ; 117(1): 51-8, 2005 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-15880581

RESUMO

We have previously reported that expression of periostin mRNA is markedly reduced in a variety of human cancer cell lines, suggesting that downregulation of periostin mRNA expression is correlated with the development of human cancers. In our study, to clarify the role of the periostin in human bladder carcinogenesis, we examined the expression of periostin mRNA in normal bladder tissues, bladder cancer tissues and bladder cancer cell lines by Northern blot analysis and RT-PCR analysis. Although the expression of periostin mRNA was detected in 100% (5/5) of normal bladder tissues, it was not detected in 3 human bladder cancer cell lines examined. It was also detected in 81.8% (9/11) of grade 1, 40.0% (4/10) of grade 2 and 33.3% (4/12) of grade 3 bladder cancer tissues, indicating that downregulation of periostin mRNA is significantly related to higher grade bladder cancer (p<0.05). To assess the tumor suppressor function of periostin, we investigated the ability of periostin gene to suppress malignant phenotypes of a bladder cancer cell line, SBT31A. Ectopic expression of periostin gene by a retrovirus vector suppressed in vitro cell invasiveness of the bladder cancer cells without affecting cell proliferation and tumor growth in nude mice. Periostin also suppressed in vivo lung metastasis of the mouse melanoma cell line, B16-F10. Mutational analysis revealed that the C-terminal region of periostin was sufficient to suppress cell invasiveness and metastasis of the cancer cells. Periostin may play a role as a suppressor of invasion and metastasis in the progression of human bladder cancers.


Assuntos
Moléculas de Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/prevenção & controle , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Animais , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/prevenção & controle , RNA Mensageiro/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/patologia
19.
Oncogene ; 23(17): 2977-87, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15021917

RESUMO

The drs gene was originally isolated as a suppressor against v-src transformation. Expression of drs mRNA was markedly downregulated in a variety of human cancer cell lines and tissues, suggesting that the drs gene acts as a tumor suppressor. In this study, we found that ectopic expression of the Drs protein induced apoptosis in human cancer cell lines. Analyses using deletion mutants of drs revealed that both the C-terminal region and the three consensus repeats in the N-terminal region are essential for the induction of apoptosis. Caspase-12, -9, and -3 were sequentially activated by drs, and specific inhibitors of caspase-3 and -9 suppressed drs-induced apoptosis. The release of cytochrome c from the mitochondria into the cytoplasm was not observed in apoptosis by drs, suggesting that the mitochondrial pathway does not mediate drs-induced apoptosis. Furthermore, we found that the Drs protein can interact with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein localized in the endoplasmic reticulum, and that coexpression of these genes increased the efficiency of apoptosis. These results indicated that Drs induces apoptosis by a novel pathway mediated by ASY/Nogo-B/RTN-x(S), caspase-12, -9, and -3.


Assuntos
Apoptose/genética , Moléculas de Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Proteínas Nucleares/genética , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células HeLa , Humanos , RNA Mensageiro/genética , Transfecção
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