RESUMO
BACKGROUND & AIMS: Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors. METHODS: We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from KrasG12D/+/Trp53R172H/+/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses. RESULTS: In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B-LYN-p85 complex, which increased PI3K-Akt-mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice. CONCLUSIONS: Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Serotonina/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Inativação Gênica , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Indóis/uso terapêutico , Ácido Láctico/biossíntese , Masculino , Camundongos , Pessoa de Meia-Idade , Monoaminoxidase/análise , Transplante de Neoplasias , Pâncreas/química , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor 5-HT2B de Serotonina/genética , Serotonina/análise , Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/uso terapêutico , Transdução de Sinais , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo , Análise Serial de Tecidos , Transcriptoma , Triptofano Hidroxilase/análise , Ureia/análogos & derivados , Ureia/uso terapêutico , Quinases da Família src/metabolismoRESUMO
The wet form of age-related macular degeneration (AMD) is a leading cause of blindness among elderly Americans and is characterized by abnormal vessel growth, termed choroidal neovascularization (CNV). Integrin α5ß1 is a transmembrane receptor that binds matrix macromolecules and proteinases to stimulate angiogenesis. We recently demonstrated that integrin α5ß1 plays a critical role in the development of choroidal neovascularization. In this study, we determined the role and underlying mechanisms of integrin α5ß1 in angiogenesis in human choroidal endothelial cells and evaluated the antiangiogenic effects of delivering a combination therapy of ATN-161, an integrin α5ß1 inhibitor, and an anti-VEGF monoclonal antibody to rats with laser-induced CNV. Vascular endothelial growth factor (VEGF) is a signaling protein that stimulates vasculogenesis and angiogenesis through a pathway that is distinct from the integrin α5ß1 signaling pathway. Our results indicate that fibronectin binds to integrin α5ß1 and synergizes VEGF-induced angiogenesis via two independent signaling pathways, FN/integrin α5ß1/FAK/ERK1/2 and FN/integrin α5ß1/FAK/AKT. Integrin α5 knockdown by shRNA inhibits endothelial cell migration, tube formation, and proliferation, while ATN-161 only partially decreases integrin α5 function. Treatment with ATN-161 combined with anti-VEGF antibody showed joint effects in attenuating angiogenesis. In summary, our results provide the first evidence for the mechanisms by which integrin α5ß1 is involved in ocular pathological neovascularization in vivo, suggesting that dual inhibition of integrin α5ß1 and VEGF may be a promising novel therapeutic strategy for CNV in wet AMD.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Integrina alfa5beta1/metabolismo , Degeneração Macular/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Fibronectinas/metabolismo , Imunofluorescência , Masculino , Ligação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
C9orf140 is a newly identified and characterized gene which is associated with cell proliferation and tumorigenicity. Expression of C9orf140 is upregulated in human gastric cancer and colorectal cancer (CRC); however, little is known about its role in CRC progression. We have investigated the clinical significance, biological effects and mechanisms of C9orf140 signaling. We found that the expression of C9orf140 is dramatically increased in a subset of CRC and correlates significantly with vascular invasion and lymph node metastasis. Our finding showed that knockdown of C9orf140 significantly reduced cell proliferation and invasion in vitro and dramatically increased overall survival and decreased lung metastasis in vivo. Conversely, overexpression of C9orf140 significantly increased lung metastasis and shortened overall survival when compared with control tumors. C9orf140-induced CRC cell invasion may depend on promoting the epithelial-mesenchymal transition progression. STAT5 may directly interact with the enhancer of zeste homolog 2 (EZH2) and ß-catenin to enhance C9orf140 gene transactivation. Furthermore, C9orf140 may participate in cell invasion which is induced by STAT5, EZH2 or ß-catenin activation. We describe the role of C9orf140 in CRC progression and find that C9orf140 overexpression may be regulated by STAT5, EZH2 and ß-catenin interaction.
Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fator de Transcrição STAT5/metabolismo , beta Catenina/metabolismo , Adulto , Idoso , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas Nucleares , Ligação Proteica , Transdução de SinaisRESUMO
BACKGROUND: A remolded microenvironment in hepatocellular carcinoma (HCC) caused by abnormally expressed matricellular proteins could promote HCC progression. The cell-matrix interactions mediated by integrins play an important role in tumor microenvironment. Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein with angiogenic and anti-inflammatory effects, is abnormally highly expressed in HCC. Here we aim to analyze its expression in liver and HCC tissues, investigate the underlined mechanisms accounted for HCC progression. METHODS: EDIL3 expression level is examined in normal liver, cirrhotic liver and HCC at both mRNA and protein level. The association between EDIL3 and clinical outcomes is analyzed. The pattern of EDIL3 expression and location is examined using Immunofluorescence and ELISA. Overexpression or knock-down of EDIL3 in a panel of cell lines are subjected to assays related to proliferation, invasion, and anoikis to investigate the mechanisms of this matrix protein in HCC progression. Recombinant EDIL3 treatment is applied to confirm the results. RESULTS: Compared with normal liver and cirrhotic liver, EDIL3 is elevated in HCC. High level of EDIL3 protein is much more commonly in patients with larger tumor or portal vein tumor thrombus (PVTT) formation, associated with poor prognosis. EDIL3 is abundantly expressed in HCC cells and secreted by cancer cells. In vitro and in vivo studies indicate that EDIL3, probably in an autocrine manner, inhibits anoikis and promotes anchorage-independent growth of HCC cells. Further mechanistic studies suggest integrin ligation by EDIL3 and thus that the sustained activation of the FAK-Src-AKT signal is responsible for the anoikis resistance and anchorage independence. Both the administration of cilengitide, a RGD-containing integrin antagonist, and silencing of integrin αV, an important RGD-binding integrin, results in the blockade of anoikis-resistance induced by EDIL3. CONCLUSION: Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of detached HCC cells and may involve in cancer cell spreading. We also highlight the importance of interaction between EDIL3 and integrin αV and suggest disrupting the ligation of EDIL3 to integrins via RGD-blocking in selected patients may bear potential therapeutic value.
Assuntos
Anoikis , Comunicação Autócrina , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Integrina alfaV/metabolismo , Neoplasias Hepáticas/patologia , Oligopeptídeos/metabolismo , Animais , Anoikis/genética , Comunicação Autócrina/genética , Proteínas de Ligação ao Cálcio , Carcinogênese/patologia , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Veia Porta/patologia , Prognóstico , Reprodutibilidade dos Testes , Transdução de Sinais , Trombose/patologia , Regulação para Cima/genéticaRESUMO
BACKGROUND & AIMS: Monoamine oxidase A (MAOA), a catecholamine neurotransmitter degrading enzyme, is closely associated with neurological and psychiatric disorders. However, its role in cancer progression remains unknown. METHODS: Hepatocellular carcinoma (HCC) tissue arrays (n=254) were used to investigate the correlation between MAOA expression and clinicopathological findings. In vitro invasion and anoikis assays, and in vivo intrahepatic and lung metastasis models were used to determine the role of MAOA in HCC metastasis. Quantitative real-time PCR, western blotting, immunohistochemical staining and HPLC analysis were performed to uncover the mechanism of MAOA in HCC. RESULTS: We found that MAOA expression was significantly downregulated in 254 clinical HCC samples and was closely correlated with cancer vasoinvasion, metastasis, and poor prognoses. We then demonstrated that MAOA suppressed norepinephrine/epinephrine (NE/E)-induced HCC invasion and anoikis inhibition, and uncovered that the effects of NE/E on HCC behaviors were primarily mediated through alpha 1A (ADRA1A) and beta 2 adrenergic receptors (ADRB2). In addition to the canonical signaling pathway, which is mediated via adrenergic receptors (ADRs), we found that ADR-mediated EGFR transactivation was also involved in NE-induced HCC invasion and anoikis inhibition. Notably, we found that MAOA could synergize with EGFR inhibitors or ADR antagonists to abrogate NE-induced HCC behaviors. CONCLUSIONS: Taken together, the results of our study may provide insights into the application of MAOA as a novel predictor of clinical outcomes and indicate that increasing MAOA expression or enzyme activity may be a new approach that can be used for HCC treatment.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Anoikis/efeitos dos fármacos , Anoikis/fisiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Linhagem Celular Transformada , Linhagem Celular Tumoral , Epinefrina/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Monoaminoxidase/genética , Norepinefrina/metabolismo , Valor Preditivo dos Testes , Prognóstico , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Dickkopf-related protein 1 (DKK1) has been reported involved in metastasis and invasion in several tumors. This study sought to investigate the prognostic value of DKK1 in intrahepatic cholangiocarcinoma (ICC) and its role in promoting ICC metastasis. METHODS: Tissue microarrays of 138 ICC patient samples were employed to detect DKK1, vascular endothelial growth factor C (VEGF-C), and matrix metalloproteinase 9 (MMP9) expression using immunohistochemistry. The prognostic significances were assessed by Kaplan-Meier survival estimates. DKK1 expression was measured in an ICC cell line (HCCC-9810) and ICC tissues by immunofluorescence assay, quantitative real-time polymerase chain reaction, and western blot. Serum levels of DKK1 from 37 ICC patients were tested by enzyme-linked immunosorbent assay. The role of DKK1 in proliferation, migration, invasion, and gene expression regulation was assessed by DKK1 depletion using small interfering RNA. RESULTS: Multivariate analyses revealed that DKK1 was an unfavorable predictor for overall survival and time to recurrence. The prognostic significance was retained in ICC patients with low recurrence risk (P < .05). DKK1 expression was elevated in an ICC cell line, tumor samples, and patient sera. High levels of DKK1 in ICC tissues correlated with elevated MMP9, VEGF-C, and metastasis of hepatic hilar lymph nodes. DKK1 depletion caused a decrease in cell migration and invasiveness, and down-regulation of MMP9 and VEGF-C expression. CONCLUSIONS: DKK1 is a novel prognostic biomarker for ICC, and it enhances tumor cell invasion and promotes lymph node metastasis of ICC through the induction of MMP9 and VEGF-C. DKK1 may be a potential therapeutic target for ICC.
Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metástase Linfática , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colangiocarcinoma/mortalidade , Colangiocarcinoma/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Homo sapiens longevity assurance homologue 2 (LASS2) is a novel gene isolated from a human liver cDNA library by our laboratory, and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene). According to our previous results, LASS2 could interact with subunit c of vacuolar type H(+)-ATPase (V-ATPase), and the overexpression of LASS2 could inhibit the cell growth of a human hepatocellular carcinoma (HCC) cell line, SMMC-7721. In order to understand the role of the interaction between LASS2 and V-ATPase in HCC cell growth, we transiently transfected plasmid pCMV-HA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The pH-sensitive fluorescence probes, BCECF and BCECF-AM, were used to measure the intracellular and extracellular H(+) concentrations of HCCLM3 cells respectively. The effect of LASS2 gene on apoptosis was evaluated with Annexin-V/FITC and propidium iodide (PI) by flow cytometry. Western blot was used to detect cytochrome c (Cyt c) in the cytosol and mitochondria, as well as pro-caspase-3 in cytosol. The results showed that the cell growth of LASS2-transfected HCCLM3 cells was significantly inhibited compared with that of the mock control. LASS2 transfection increased intracellular H(+) concentration of HCCLM3 cells, while decreased extracellular H(+) concentration. Moreover, LASS2 transfection significantly enhanced the apoptosis of HCCLM3 cells. In LASS2-transfected cells, the amounts of Cyt c increased in the cytosol, while decreased in the mitochondria. Meanwhile, the expression of pro-caspase-3 in the cytosolic extracts was decreased. These results implicate that LASS2 gene might increase intracellular H(+) of HCC cells via the interaction with V-ATPase, thereby inducing cell apoptosis through mitochondrial pathway.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA Interferente Pequeno , TransfecçãoRESUMO
BACKGROUND: Dickkopf-1 (DKK1), a secreted protein, is known as a negative regulator of the Wnt signaling pathway, which has been implicated in the development of several types of cancers. Clinical significance of serum DKK1 in lung cancer remains to be determined. METHODS: A novel time-resolved immunofluorometric assay was developed. By use of this method, we investigated the serum concentrations of DKK1 in 592 patients with malignancies, 72 patients with benign lung disease, and 120 healthy controls. Serum cytokeratin 19 fragment and neuron-specific enolase values were obtained. RESULTS: Serum DKK1 concentrations were significantly higher in patients with lung cancer than in patients with other malignant tumors or benign lung diseases and healthy controls. Serum concentrations of DKK1 were decreased significantly in groups of patients with gastric cancer, colorectal cancer, ovarian cancer, and cervical adenocarcinoma compared with healthy controls. Application of both DKK1 and cytokeratin 19 fragment increased sensitivity, correctly identifying 89.6% of the non-small cell lung cancer patients as positive. The use of both DKK1 and neuron-specific enolase increased sensitivity to detect small cell lung cancer to 86.2%. DKK1 concentrations increased with stage, tumor class, and presence of lymph node and distant metastases, regardless of histology and patient age and sex. Patients with a DKK1 concentration of 22.6 microg/L or higher had a statistically significantly diminished survival compared with patients whose DKK1 values were lower. CONCLUSIONS: DKK1 was preferentially expressed in lung cancer. Increasing concentrations of DKK1were significantly associated with tumor progression and decreased survival in patients with lung cancer. .
Assuntos
Biomarcadores Tumorais/sangue , Fluorimunoensaio/métodos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Neoplasias Pulmonares/sangue , Idoso , Calibragem , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Taxa de SobrevidaRESUMO
Alpha-fetoprotein (AFP) is a commonly used tumor marker in the detection of hepatocellular carcinoma (HCC), and its sensitivity and specificity is insufficient to detect HCC in all patient samples. Recently, the immunocomplexed forms of AFP (AFP-IgM) have been reported to be present in HCC patients. This study was aimed at developing a novel time-resolved immunofluorometric assay (TR-IFMA) for the simultaneous determination of AFP-IgM and AFP concentration in HCC. We constructed a double-label assay by using Sm3+-labeled mAb to human IgM antibody, Eu3+-labeled mAb to AFP, and immobilized another mAb to AFP on the solid phase. The performances of the assay were all found to be satisfactory. The validity of the novel assay was confirmed by the good correlation between the results obtained by TR-IFMA and commercial ELISA or electrochemiluminescence immunoassay. AFP-IgM and AFP were increased above the cutoffs in 65.25 and 45.76% of HCC, respectively. ROC analysis yielded the following area under the curve: AFP-IgM 0.774 (CI 95% 0.736-0.809), AFP 0.771 (CI 95% 0.733-0.860). The combined use of AFP-IgM and AFP increased the sensitivity of detection to 72.88% in patients with HCC. These data suggest that the use of a combination of two markers in clinical practice could increase the accuracy of HCC diagnosis.
Assuntos
Complexo Antígeno-Anticorpo/análise , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Fluorimunoensaio/métodos , Neoplasias Hepáticas/sangue , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/imunologia , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/imunologia , Biomarcadores Tumorais/imunologia , Calibragem , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/imunologia , Ensaio de Imunoadsorção Enzimática , Hepatite Crônica/sangue , Humanos , Imunoglobulina M/imunologia , Cirrose Hepática/sangue , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/imunologia , Valor Preditivo dos Testes , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Understanding mechanisms of cancer metastasis is crucial for reduction of cancer mortality. Acyl-CoA medium-chain synthetase 3 (ACSM3) is an acyl-CoA synthetase which takes part in the first step of fatty acid metabolism. However, the expression, clinical significance and biological function of ACSM3 remain unknown in hepatocellular carcinoma (HCC). In this study, the expression and prognostic relevance of ACSM3 were investigated by tissue microarray and HCC clinical samples. Migration and invasion assays were carried out for functional analysis in vitro and a xenograft model was used to analyze the effects of ACSM3 on cancer metastasis in vivo. Furthermore, human phospho-kinase array assays were performed to explore molecular mechanisms of ACSM3 in HCC. The results showed ACSM3 was downregulated in HCC tissues. HCC patients with low expression of ACSM3 exhibited poor prognosis. Overexpression of ACSM3 attenuated migration and invasion of HCC cells in vitro and in vivo and downregulated the phosphorylation of WNK1 and AKT. Our findings indicate ACSM3 is a novel prognostic marker and a potential therapeutic target for HCC.
RESUMO
Collagen triple helix repeats containing 1 (CTHRC1) participates in vascular remodeling, bone formation, and developmental morphogenesis. Recently, CTHRC1 has been found up-regulated in many solid tumors and contributes to tumorigenesis, but its role in the progression of human colorectal cancer (CRC), remains unclear. In this study, CTHRC1 expression in human CRC cell lines was evaluated by quantitative real-time PCR and immunoblot analyses. The role of CTHRC1 in CRC cell proliferation and extracellular matrix invasion in vitro was analyzed by gene over-expression and recombinant protein. Reporter luciferase assay was used to reveal key relevant signaling pathways involved in CRC cells. The results show that CTHRC1 is secreted both by colorectal epithelia cells and stromal fibroblasts. Recombinant CTHRC1 promotes CRC cell migration and invasion dose-dependently. CTHRC1 overexpression promotes CRC cell migration, invasion and proliferation in vitro. Wnt/PCP signaling but not Wnt/catenin signaling was activates by CTHRC1 in CRC cells. Together, CTHRC1 promotes CRC cell proliferation, migration and invasion in vitro, which is possibly mediated by activating Wnt/PCP pathway.
Assuntos
Proliferação de Células/genética , Neoplasias Colorretais/genética , Proteínas da Matriz Extracelular/genética , Invasividade Neoplásica/genética , Transdução de Sinais/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Movimento Celular/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/patologiaRESUMO
BACKGROUND: Accumulating evidence links colorectal cancer (CRC) with the intestinal microbiota. However, the disturbance of intestinal microbiota and the role of Fusobacterium nucleatum during the colorectal adenoma-carcinoma sequence have not yet been evaluated. METHODS: 454 FLX pyrosequencing was used to evaluate the disturbance of intestinal microbiota during the adenoma-carcinoma sequence pathway of CRC. Intestinal microbiota and mucosa tumor-immune cytokines were detected in mice after introducing 1,2-dimethylhydrazine (DMH), F. nucleatum or Berberine (BBR), using pyrosequencing and Bio-Plex Pro™ cytokine assays, respectively. Protein expressions were detected by western blotting. RESULTS: The levels of opportunistic pathogens, such as Fusobacterium, Streptococcus and Enterococcus spp. gradually increased during the colorectal adenoma-carcinoma sequence in human fecal and mucosal samples. F. nucleatum treatment significantly altered lumen microbial structures, with increased Tenericutes and Verrucomicrobia (opportunistic pathogens) (P < 0.05 = in wild-type C57BL/6 and mice with DMH treatment). BBR intervention reversed the F. nucleatum-mediated increase in opportunistic pathogens, and the secretion of IL-21/22/31, CD40L and the expression of p-STAT3, p-STAT5 and p-ERK1/2 in mice, compared with mice fed with F. nucleatum alone. CONCLUSIONS: F. nucleatum colonization in the intestine may prompt colorectal tumorigenesis. BBR could rescue F. nucleatum-induced colorectal tumorigenesis by modulating the tumor microenvironment and blocking the activation of tumorigenesis-related pathways.
Assuntos
Adenoma/prevenção & controle , Berberina/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Colorretais/prevenção & controle , Infecções por Fusobacterium/complicações , Fusobacterium nucleatum/patogenicidade , Microambiente Tumoral/efeitos dos fármacos , Adenoma/etiologia , Adenoma/patologia , Animais , Western Blotting , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Citocinas/genética , Citocinas/metabolismo , Fezes/microbiologia , Infecções por Fusobacterium/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
AIMS: To investigate the antitumor effects of probiotics Clostridium butyricum and Bacillus subtilis on colorectal cancer (CRC) progression. MATERIALS & METHODS: The effects of C. butyricum and B. subtilis on CRC cells were studied. Male C57BL/6 mice with 1,2-dimethylhydrazine dihydrochloride (DMH)-induced CRC were intervened by these two probiotics and the antitumor effects were examined by comparing the tumor incidence and detecting the inflammatory and immune-related markers. RESULTS & CONCLUSIONS: C. butyricum and B. subtilis inhibited the proliferation of CRC cells, caused cell cycle arrest and promoted apoptosis. In vivo, these two probiotics inhibited the development of DMH-induced CRC. The molecular mechanism involved reduced inflammation and improved immune homeostasis. This work establishes a basis for the protective role of probiotics B. subtilis and C. butyricum in intestinal tumorigenesis.
Assuntos
Bacillus subtilis/fisiologia , Carcinogênese , Clostridium butyricum/fisiologia , Neoplasias Colorretais/prevenção & controle , Probióticos , 1,2-Dimetilidrazina , Administração Oral , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/induzido quimicamente , Meios de Cultivo Condicionados , Progressão da Doença , Homeostase , Inflamação/terapia , Masculino , Camundongos Endogâmicos C57BLRESUMO
hNRAGE, a neurotrophin receptor p75 interacting MAGE homologue, is cloned from a human placenta cDNA library. hNRAGE can inhibit the colony formation of and arrest cell proliferation at the G1/S and G2/M stages in hNRAGE overexpressing cells. Interestingly, hNRAGE also increases the p53 protein level as well as its phosphorylation (Ser392). Further studies demonstrated that hNRAGE does not affect the proliferation of mouse p53-/- embryonic fibroblasts, suggesting that p53 function is required for hNRAGE induced cell cycle arrest. Moreover, the cell cycle inhibiting protein p21(WAF) is induced by hNRAGE in a p53 dependent manner. The data provide original evidence that hNRAGE arrests cell growth through a p53 dependent pathway.
Assuntos
Interfase , Proteínas de Neoplasias/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , Antígenos de Neoplasias , Divisão Celular , Linhagem Celular Tumoral , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Regulação da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No.AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431 and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellular carcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate that IC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Transporte/isolamento & purificação , Divisão Celular/genética , Transformação Celular Neoplásica/genética , Quinases Ciclina-Dependentes/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Cromossomos Humanos Par 17 , Clonagem Molecular , Quinase 5 Dependente de Ciclina , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Ligação Proteica/genética , Isoformas de Proteínas/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transfecção , Proteínas Supressoras de TumorRESUMO
CT120, a novel membrane-associated gene implicated in lung carcinogenesis, was previously identified from chromosome 17p13.3 locus, a hot mutation spot involved in human malignancies. In the present study, we further determined that CT120 ectopic expression could promote cell proliferation activity of NIH3T3 cells using MTS assay, and monitored the downstream effects of CT120 in NIH3T3 cells with Atlas mouse cDNA expression arrays. Among 588 known genes, 133 genes were found to be upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120: One is the Raf/MEK/Erk signal cascades and the other is the PI3K/Akt signal cascades, suggesting that CT120 might contribute, at least in part, to the constitutively activation of Erk and Akt in human lung cancer cells. In addition, some tumor metastasis associated genes cathepsin B, cathepsin D, cathepsin L, MMP-2/TIMP-2 were also upregulated by CT120, upon which CT120 might be involved in tumor invasiveness and metastasis. In addition, CT120 might play an important role in tumor progression through modulating the expression of some candidate "Lung Tumor Progression" genes including B-Raf, Rab-2, BAX, BAG-1, YB-1, and Cdc42.
Assuntos
Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Animais , Proliferação de Células , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
AIM: Bcl-2/adenovirus E1B 19 ku interacting protein 2-like (BNIPL-2) is a novel protein recently identified in our laboratory. BNIPL-2 is homologous to human BNIP-2, a potentially proapoptotic protein, and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells. Here we report the gene-expression profile regulated by BNIPL-2 in human hepatocarcinoma Hep3B cells and the analysis of its potential roles in cell apoptosis. METHODS: BNIPL-2 was overexpressed in Hep3B cells using tetracycline inducible or Tet-on system. Screened by Western blot, the cells with low background and high induction fold of BNIPL-2 were obtained. We performed Atlas human cDNA expression array hybridization on these cells and analyzed the data with Quantarray software to identify BNIPL-2-regulated genes and their expression profile. RT-PCR was used to confirm the altered expression level of part of genes identified by the Atlas array hybridization. RESULTS: Fifteen of 588 genes spotted on the Atlas membrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells, in which 8 genes involved in cell apoptosis or growth inhibition were up-regulated and 7 genes involved in cellular proliferation were down-regulated following overexpression of BNIPL-2. CONCLUSION: cDNA array is a powerful tool to explore gene expression profiles under inducible conditions. The data obtained using the cDNA expression microarray technology indicates that BNIPL-2 may play its roles in apoptosis through regulating the expression of genes associated with cell apoptosis, growth inhibition and cell proliferation.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
OBJECTIVE: To develop a DNA vector-based RNA interference (RNAi) technology that inhibits the activity of the telomerase of the hepatocellular carcinoma cell line HCCLM3 in order to suppress the proliferation of the cells. METHODS: Hepatocellular carcinoma cells of the line HCCLM3 were cultured. mRNA interfering double-stranded DNA vector PSG-AS targeting the mRNA of human telomerase reverse transcriptase (hTERT) and the control vector PSG-CTR were constructed respectively, and then were transfected into the HCCLM3 cells. The expression of hTERT of the transfected cells was determined by Western blotting and the activity of telomerase was determined by telomeric repeat amplification-ELISA (TRAP-ELISA). Flow cytometry was used to detect the apoptosis of transfected cells and MTS method was used to measure the growth curve of the cells so as to observe the effect of the PSG-AS on the proliferation of HCCLM3 cell. RESULTS: TRAP-ELISA showed that the inhibition rate of PSG-AS on the telomerase activity was 76%. The apoptotic rate of the PSG-AS group was significantly higher than that of the PSG-CTR group (t = 11.48, P < 0.001). Western blotting showed a remarkable inhibition of hTERT protein in the PSG-AS group. CONCLUSION: Capable of suppressing the hTERT expression and the activity of telomerase, RNA interfering technology can be applied to treatment of tumors.
Assuntos
Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Interferência de RNA , RNA Neoplásico/metabolismo , Telomerase/metabolismo , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Divisão Celular , Linhagem Celular Tumoral , Reparo do DNA , Proteínas de Ligação a DNA , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/genética , TransfecçãoRESUMO
OBJECTIVE: Previous studies related to the prognostic value of E-Cadherin expression on non-small cell lung cancer (NSCLC) were inconsistent. The present study aimed to evaluate the relation between E-Cadherin expression and the prognosis of NSCLC. METHODS: We performed a meta-analysis based on 14 studies including 2395 NSCLC patients. Literature retrieval, data extraction, and meta-analyses were performed according to the Revman 5.0 guidelines. We utilized the fixed-effect model to pool the HR according to the test of heterogeneity in the meta-analysis. RESULTS: A total of 14 eligible studies including 2395 NSCLC patients were analyzed. In total, 51.2% of the patients were considered as having reduced expression of E-Cadherin according to the authors' cutoff. The pooled hazard ratio (HR) of reduced expression of E-Cadherin for overall survival (OS) was 1.19 (95% CI: 1.01 to 1.40, P=0.04), showing a worse survival when E-Cadherin expression is decreased. CONCLUSION: Patients with reduced expression of E-cadherin have a poorer OS compared with those with normal or high expression of E-cadherin.
RESUMO
Paraneoplastic Ma1 (PNMA1) is a member of an expanding family of 'brain/testis' proteins involved in an autoimmune disorder defined as paraneoplastic neurological syndrome (PNS). Although it is widely studied in PNS, little is known about the underlying clinical significance and biological function of PNMA1 in tumors. Here, we find that elevated PNMA1 expression is more commonly observed in pancreatic ductal adenocarcinoma (PDAC) cell lines, compared with normal pancreatic cell and tissues from pancreatic ductal adenocarcinoma patient. Besides, higher PNMA1 expression is closely correlated with large tumor size. Suppression of endogenous PNMA1 expression decreases cell viability and promotes cell apoptosis. Subsequent studies reveal that the PI3K/AKT, MAPK/ERK pathway and members of the anti-apoptotic Bcl-2 family may be involved in the pro-survival and anti-apoptotic effect of PNMA1 on PDAC. Taken together, this study provides evidence that PNMA1 is involved in tumor growth of pancreatic carcinoma and PNMA1-related pathways might represent a new treatment strategy.