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1.
Onco Targets Ther ; 15: 291-298, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35345394

RESUMO

Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world with a five-year survival rate of less than 20%. Nonetheless, selecting an appropriate therapeutic agent to inhibit the development of hepatoma cells is still a challenge. Bufalin, a component of the traditional Chinese medicine Chansu, has been shown to inhibit the proliferation, invasion and metastasis of HCC through various signaling pathways. In addition, bufalin and sorafenib demonstrate a synergistic effect in cancer therapeutics. This review highlighted on several focal signaling pathways involved in the inhibitory effects of bufalin on HCC and its synergistic mechanisms with sorafenib. The immunotherapy effect of bufalin has also been discussed as a novel property.

2.
Zhongguo Zhong Yao Za Zhi ; 44(3): 475-481, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30989911

RESUMO

In this study, taking Cistanche deserticola in Xinjiang as the experimental material, the optimal process for extracting polysaccharides from C. deserticola with water extraction was studied by using single factor and orthogonal experiment. Its effects on protein removal and polysaccharides retaining were investigated by using Sevag, enzymatic method or combination of these two methods, so as to determine the optimal method for protein removal from polysaccharides of C. deserticola; the decolorization and purification methods such as macroporous resin of AB-8 and activated Carbon were used to determine the optimal process. The results showed that the extraction rate of polysaccharides from C. deserticola was 18.40% during the optimal process of the water extraction as follows: extraction temperature 75 ℃, extraction time 165 min and solid-liquid ratio 1∶55. The protein removal rate can reach 31.40% and polysaccharide retention rate can reach 96.00% under the optimal protein removal process: temperature 50 ℃, time 2 h, and papain dosage 0.2%. The decolorization rate of activated Carbon and macroporous resin called AB-8 was 80.37% and 86.43%, and the recovery rate of polysaccharides was 77.05% and 91.93%, respectively, suggesting that macroporous resin was more suitable for decoloration. Macroporous resin named AB-8 increased the purity of the polysaccharide crude extract from 67.70% to 84.80% under the following conditions: concentration of the sample 4 g·L~(-1), concentration of the eluent 60% ethanol, and the flow rate 1 mL·min~(-1), showing significant purification effect.


Assuntos
Cistanche/química , Extratos Vegetais/química , Polissacarídeos/isolamento & purificação , Temperatura , Água
3.
Cell Death Dis ; 9(11): 1120, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389909

RESUMO

Estrogen receptor ß (ERß) plays critical roles in thyroid cancer progression. However, its role in thyroid cancer stem cell maintenance remains elusive. Here, we report that ERß is overexpressed in papillary thyroid cancer stem cells (PTCSCs), whereas ablation of ERß decreases stemness-related factors expression, diminishes ALDH+ cell populations, and suppresses sphere formation ability and tumor growth. Screening estrogen-responsive lncRNAs in PTC spheroid cells, we find that lncRNA-H19 is highly expressed in PTCSCs and PTC tissue specimens, which is correlated with poor overall survival. Mechanistically, estradiol (E2) significantly promotes H19 transcription via ERß and elevates H19 expression. Silencing of H19 inhibits E2-induced sphere formation ability. Furthermore, H19 acting as a competitive endogenous RNA sequesters miRNA-3126-5p to reciprocally release ERß expression. ERß depletion reverses H19-induced stem-like properties upon E2 treatment. Appropriately, ERß is upregulated in PTC tissue specimens. Notably, aspirin attenuates E2-induced cancer stem-like traits through decreasing both H19 and ERß expression. Collectively, our findings reveal that ERß-H19 positive feedback loop has a compelling role in PTCSC maintenance under E2 treatment and provides a potential therapeutic targeting strategy for PTC.


Assuntos
Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Antineoplásicos/farmacologia , Aspirina/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Receptor beta de Estrogênio/metabolismo , Retroalimentação Fisiológica , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Análise de Sobrevida , Câncer Papilífero da Tireoide/tratamento farmacológico , Câncer Papilífero da Tireoide/mortalidade , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/mortalidade , Neoplasias da Glândula Tireoide/patologia
4.
Biomed Res Int ; 2015: 153987, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26221582

RESUMO

OBJECTIVE: To investigate the effects of ß-Elemene (ß-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. METHODS: After treatment with ß-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of ß-ELE on DNA breaks. RESULTS: ß-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. ß-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. ß-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. CONCLUSION: ß-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.


Assuntos
Antígenos de Neoplasias/biossíntese , Carcinoma Hepatocelular/genética , DNA Topoisomerases Tipo II/biossíntese , DNA Topoisomerases Tipo I/biossíntese , Proteínas de Ligação a DNA/biossíntese , Neoplasias Hepáticas/genética , Sesquiterpenos/administração & dosagem , Antígenos de Neoplasias/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia
5.
PLoS One ; 10(5): e0125924, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25978404

RESUMO

BACKGROUND: Prostate-specific membrane antigen (PSMA) has been found in tumor neovasculature endothelial cells (NECs) of non-prostate cancers and may become the most promising target for anti-tumor therapy. To study the value of PSMA as a potential new target for lung cancer treatment, PSMA expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and its relationship with clinicopathology were investigated in the current study. METHODS: Immunohistochemistry was used to detect PSMA expression in a total of 150 lung specimens of patients with lung cancer. The data were analyzed using univariate and multivariate statistical analyses. RESULTS: The percentages of NSCLC patients who had PSMA (+) tumor cells and PSMA (+) NECs were 54.02% and 85.06%, respectively. The percentage of patients younger than 60 years old who had PSMA (+) tumor cells was 69.05%, which was significantly greater than the percentage of patients aged 60 years or older (40.00%, p<0.05). A significant difference was observed in the percentage of NSCLC patients with PMSA (+) NECs and stage I or II cancer (92.98%) and those patients with stage III or IV cancer (76.77%). In the SCLC tissues, NEC PSMA expression (70.00%) did not differ significantly from NSCLC. SCLC tumor cells and normal lung tissues cells were all negative. There was no significant correlation between the presence of PSMA (+) NECs in SCLC patients and the observed clinicopathological parameters. CONCLUSIONS: PSMA is expressed not only in NECs of NSCLC and SCLC but also in tumor cells of most NSCLC patients. The presence of PSMA (+) tumor cells and PSMA (+) NECs in NSCLC was negatively correlated with age and the clinicopathological stage of the patients, respectively.


Assuntos
Antígenos de Superfície/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glutamato Carboxipeptidase II/genética , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Adulto , Idoso , Antígenos de Superfície/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica , Carcinoma de Pequenas Células do Pulmão/metabolismo
6.
Mol Med Rep ; 12(1): 1598-604, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25815590

RESUMO

The present study aimed to investigate the effects of cinobufacini injection on the proliferation and expression of topoisomerases in human HepG-2 hepatocarcinoma cells. The cells were divided into a control group and an experimental group, in which 0.105, 0.21, 0.42 mg/l cinobufacini was injected. Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay, levels of apoptosis were detected using annexin V/propidium iodide staining and cell cycles were analyzed using flow cytometric analysis. The mRNA and protein expression levels of topoisomerase (TOPO) I and TOPO II were determined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Cinobufacini injection significantly inhibited the proliferation of the HepG-2 cells (P<0.05), induced apoptosis (P<0.05) in a dose- and time-dependent manner, induced tumor cell arrest at the S phase in a dose-dependent manner, and downregulated the mRNA and protein expression levels of TOPO I and TOPO II (P<0.05) in a dose-dependent manner. Therefore, cinobufacini was found to inhibit human HepG-2 hepatocellular carcinoma cell proliferation, and downregulation of the expression levels of TOPO I and TOPO II may contribute to the effect on proliferation observed in the Hep­G2 cells following cinobbufacini injection.


Assuntos
Carcinoma Hepatocelular/genética , DNA Topoisomerases Tipo II/biossíntese , DNA Topoisomerases Tipo I/biossíntese , Neoplasias Hepáticas/genética , Venenos de Anfíbios/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , RNA Mensageiro/biossíntese
7.
World J Gastroenterol ; 15(48): 6117-22, 2009 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-20027687

RESUMO

AIM: To investigate the possible correlation between osteoglycin expression and gelatinase activity of mouse hepatocarcinoma Hca-F cells. METHODS: A eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was constructed and transfected into Hca-F cells to investigate the possible correlation between osteoglycin expression and gelatinase activity of Hca-F cells cultured with extract of lymph node, liver, spleen or in DMEM medium. The activity of gelatinases was examined through zymographic analysis. RESULTS: High expression of osteoglycin attenuated the gelatinase activity of Hca-F cells cultured with extract of lymph node, and at the same time, decreased the metastatic potential of Hca-F cells to peripheral lymph nodes in vivo. CONCLUSION: High expression of osteoglycin decreases the gelatinase activity of Hca-F cells cultured with extract of lymph node; regulation of gelatinase activity might be one of mechanisms that osteoglycin contributes to lymphatic metastasis suppression.


Assuntos
Carcinoma Hepatocelular/enzimologia , Gelatinases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/enzimologia , Animais , Linhagem Celular Tumoral , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Transfecção
8.
Epilepsia ; 50(12): 2638-47, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19674059

RESUMO

PURPOSE: A prolonged seizure, status epileptics (SE), is a potent stimulus for increased neurogenesis in the dentate gyrus of the hippocampus. Molecular mechanisms that regulate normal and pathologic cell birth in the dentate gyrus are poorly understood. METHODS: Lithium-pilocarpine was used to induce SE in immature postnatal day 20 rats. Newborn cells in the dentate were labeled with bromo-deoxyuridine to determine a time-course of cell proliferation, and measure cell-cycle length. In addition, we studied expression by Western blot and immunohistochemistry of two known inhibitors of G(1)-S cell-cycle progression P27/Kip1 and P15/Ink4b following SE. RESULTS: Cell proliferation in the dentate gyrus increases starting 2 h after SE and is sustained for 40 days. Increased cell proliferation following SE is associated with a shortened dentate gyrus progenitor's cell cycle, 15 h in control to 12 h in the SE animals. To identify molecules responsible for the shortened progenitor cell cycle we studied inhibitors of cell-cycle progression P27/Kip1, and P15/Ink4b. We find decreased phosphorylation at P27/Kip1 Serine 10 and Threonine 187 following SE. Although total P27/Kip1 and P15/Ink4b levels were not altered after SE, P27/Kip1 immunoreactivity was minimal in newborn but increased with maturation of the dentate granule neurons. DISCUSSION: The sustained increase in dentate gyrus cell proliferation following SE provides a large pool of immature dentate granule cells prior to development of spontaneous seizures. A decrease in cell-cycle length of dentate gyrus progenitors is at least partially responsible for increased numbers of newborn cells following SE.


Assuntos
Giro Denteado/fisiologia , Estado Epiléptico/fisiopatologia , Animais , Animais Recém-Nascidos , Ciclo Celular/fisiologia , Proliferação de Células , Hipocampo , Humanos , Imuno-Histoquímica , Lítio , Neurônios , Pilocarpina , Células-Tronco/fisiologia
9.
World J Gastroenterol ; 12(42): 6893-7, 2006 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-17106944

RESUMO

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho-genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Hepáticas/genética , Metástase Linfática/genética , Animais , Ascite/patologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Perfilação da Expressão Gênica , Hibridização Genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/genética , Supressão Genética
10.
Eur J Neurosci ; 23(11): 2857-63, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16819974

RESUMO

There is an increase in the birth of dentate granule neurons after status epilepticus (SE) and there are concurrent alterations in neurotransmitter receptor expression that may contribute to the development of spontaneous seizures. To determine whether newborn and/or mature dentate granule neurons have altered neurotransmitter receptor expression after SE, we dissected individual immature, PSA-NCAM-expressing, or mature, NeuN-expressing, dentate granule neurons 2 weeks after lithium-pilocarpine-induced SE in postnatal day 20 rats. Amplified single-cell RNA was used to probe reverse Northern blots containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate neurotransmitter receptor subunits. Two weeks after lithium-pilocarpine-induced SE there were increases in AMPA GluR2 and kainate KA2 subunit mRNA and decreases in AMPA GluR3 and kainate GluR6 receptor subunit mRNA levels in mature dentate granule neurons. In contrast, only the kainate GluR6 subunit expression was reduced in immature dentate granule neurons after SE. Alterations in transcription of excitatory amino acid receptor subunits after SE occur primarily in the mature population of dentate granule neurons. Our findings suggest that neurotransmitter receptor gene expression is altered differently in immature and mature dentate granule neurons following SE, and may result in differential contributions of these two groups of dentate granule neurons to the subsequent development of epilepsy.


Assuntos
Giro Denteado , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Estado Epiléptico/fisiopatologia , Animais , Animais Recém-Nascidos , Northern Blotting/métodos , Western Blotting/métodos , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/patologia , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Imuno-Histoquímica/métodos , Lítio/toxicidade , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Fosfopiruvato Hidratase/metabolismo , Pilocarpina/toxicidade , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de Ácido Caínico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ácidos Siálicos/metabolismo , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia
11.
Ai Zheng ; 24(7): 774-80, 2005 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-16004799

RESUMO

BACKGROUND & OBJECTIVE: Metastasis is a complex process involving multiple genetic changes. The high mortality and poor prognosis caused by metastasis in malignant tumor patients and the uncertain mechanisms are always the prominent problems in the field of oncology. In order to screen for lymphatic metastasis-associated genes, the gene expression profiles of mouse hepatocarcinoma cell lines Hca-F (highly metastatic) and Hca-P (low metastatic) were compared by gene chip. METHODS: Total RNA was isolated from Hca-F and Hca-P cells, and synthesized into double-stranded cDNA, then synthesized into biotin-labeled cRNA probes by in vitro transcription. The cRNA probes were separately hybridized with Affymetrix GeneChip MOE430A (containing 22,690 transcripts, including 14,500 known mouse genes and 4,371 ESTs), and the signals were scanned by the GeneArray Scanner. The results were analyzed by bioinformatics. RESULTS: Compared with the gene expression profile of Hca-P cells, 901 (6.2%) genes and 129 (3%) ESTs were up-regulated by at least 2 folds in Hca-F cells; 33 genes, including endoglin (EDG; CD105), Mcam (Muc18; Mel-CAM; CD146), Cdc42ep5 (CEP5; Borg3), Ptprr (protein tyrosine phosphatase, receptor type, R), F2r [coagulation factor II (thrombin) receptor; Par1; ThrR], D7Ertd458e (necl-5), NR1D1, Serpin h1 (HSP47), AXL, Mak, and Areg (AR), were up-regulated 13.93-29.86 folds. According to Gene Ontology and Treeview analysis, these 33 genes were involved in angiogenesis, cell adhesion, signal transduction, cell motility, transcription, chaperone activity, protein kinase activity, receptor binding, and so on. CONCLUSION: Many lymphatic metastasis-associated genes were screened by high-throughput gene chip method; validating their cellular functions will help to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.


Assuntos
Perfilação da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Metástase Linfática/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Linhagem Celular Tumoral , Camundongos
12.
Zhonghua Zhong Liu Za Zhi ; 27(3): 138-40, 2005 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-15946560

RESUMO

OBJECTIVE: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Ten differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones. CONCLUSION: SSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.


Assuntos
Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Metástase Linfática/genética , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/genética
13.
World J Gastroenterol ; 11(12): 1837-42, 2005 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-15793877

RESUMO

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Testes Genéticos/métodos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/secundário , Metástase Linfática/genética , Animais , Ascite/genética , Linhagem Celular Tumoral , Desoxirribonucleases de Sítio Específico do Tipo II , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Linfonodos/patologia , Camundongos , Transplante de Neoplasias , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Neoplásico/análise
14.
World J Gastroenterol ; 11(10): 1463-72, 2005 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-15770722

RESUMO

AIM: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential. METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip(r) MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics. RESULTS: Out of the 14 500 known genes investigated, 110 (0.8%) were up regulated at least 2(3) fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 2(3) fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile. CONCLUSION: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Metástase Linfática/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Camundongos
15.
World J Gastroenterol ; 10(16): 2318-22, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15285011

RESUMO

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.


Assuntos
Carcinoma Hepatocelular/genética , DNA Complementar/genética , Neoplasias Hepáticas/genética , Metástase Linfática/genética , Animais , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Mapeamento Cromossômico , Clonagem Molecular , Biblioteca Gênica , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Neoplásico/genética
16.
J Biol Chem ; 279(20): 21012-20, 2004 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-15024013

RESUMO

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.


Assuntos
Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Substituição de Aminoácidos , Biotinilação , Linhagem Celular , DNA Complementar/genética , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Glicosilação , Humanos , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/química , Microscopia Confocal , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tunicamicina/farmacologia
17.
J Neurosci Methods ; 131(1-2): 27-33, 2003 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-14659820

RESUMO

Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 degrees C. When there is sufficient Na+ in the medium (> or =25 mM), [3H]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 degrees C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, D-amphetamine, p-tyramine, and DL-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 degrees C.


Assuntos
Cocaína/análogos & derivados , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso , Ensaio Radioligante/métodos , Sódio/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Linhagem Celular , Cocaína/antagonistas & inibidores , Cocaína/química , Cocaína/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/química , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Humanos , Rim/citologia , Nortropanos/farmacocinética , Temperatura , Trítio/farmacocinética
18.
Naunyn Schmiedebergs Arch Pharmacol ; 365(4): 303-11, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11919655

RESUMO

The role of Na(+) in the recognition of blockers by the dopamine transporter is accomodated by a model with a cation site that overlaps with the blocker binding domain, and a distal Na(+) site that interacts with this cation site and perhaps with the blocker binding domain itself. The present study addresses the application of this model to the recognition of substrates by the dopamine transporter, focusing on conditions that should reveal a stimulatory effect, if present, of Na(+) on substrate binding. Recognition was studied via the inhibition of binding of [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to the human dopamine transporter in human embryonic kidney 293 cells. Little or no changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-), both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and d-amphetamine showed binding increases upon raising Na(+), leveling off with NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+), consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and, with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed. This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50) values in Na(+) free buffer were not lower for dopamine and d-amphetamine than p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+) disinhibition. All results together support the existence of a substrate domain overlapping with a cation site that also binds Tris(+); a distal Na(+) site interacts with this cation site and with the substrate domain by negative allosterism and is additionally impacted by Cl(-). Importantly, interactions between sites vary with the type of substrate, and, in membrane preparations, Na(+) is not required for, or stimulatory to, the binding of any of the four substrates studied unlike the binding of the cocaine analog WIN 35,428.


Assuntos
Anfetamina/metabolismo , Cocaína/análogos & derivados , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso , Octopamina/metabolismo , Sódio/metabolismo , Tiramina/metabolismo , Ligação Competitiva , Linhagem Celular , Cocaína/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Rim/citologia , Especificidade por Substrato
19.
Ai Zheng ; 21(11): 1192-6, 2002 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-12526214

RESUMO

BACKGROUND & OBJECTIVE: Metastasis is the leading cause of tumor-related death, in which lymphogenous metastasis is the most common pattern and also a bridgehead of further metastasis. However, the molecular mechanism of metastasis is uncertain. This study was designed to investigate the correlation between the metastasis of hepatocarcinoma cell(HCC) to lymphnode and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymphnodes. METHODS: The maps of MMPs in supernatants of mouse HCCs with different metastatic potential Hca-F(high potential) and Hca-P cells(low potential) cultured with extract of lymph node, liver or spleen were examined by zymographic analysis. Growth fraction of lymphocytes in lymph nodes of tumor burden mice was detected by flow cytometry. The apoptosis signals of macrophages in lymph nodes were observed with TUNEL staining. The expressions of Fas ligand of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. RESULTS: With the presence of the extraction of lymph note, the quantity of MMP-9 significantly increased (P < 0.01): in RPMI1640 medium, the quantity of MMP-9 produced by Hca-F and Hca-P were 1256 +/- 157 and 2642 +/- 385, respectively. After the addition of extraction of lymph node, the quantity of MMP-9 increased to 12,403 +/- 894 and 9086 +/- 686, respectively, together with the secretion of a large amount of MMP-2 (Hca-F: 7364 +/- 2001, Hca-P: 2997 +/- 1990) and active MMP-9 (Hca-F: 7297 +/- 1657, Hca-P: 3914 +/- 1253), and the amount of which was also more in Hca-F than in Hca-P cells(P < 0.05). There was no MMPs detected when the extraction of liver and spleen is present. The growth fraction of lymphocytes was as follow: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post-inoculation and then decreased rapidly, while in the Hca-P cells, the peak appeared on the 7th day post-inoculation and then kept at a high level. TUNEL staining showed that the positive signals of macrophages were detected around Hca-F cells. The expression of Fas ligand of Hca-F cells was stronger than that of Hca-P cells (P < 0.01). CONCLUSION: The MMPs secretion of Hca-F and Hca-P cell depend on the environment of lymph nodes. The increased expression of Fas ligand protein in Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.


Assuntos
Neoplasias Hepáticas Experimentais/enzimologia , Linfonodos/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Apoptose , Proteína Ligante Fas , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Metástase Linfática , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Células Tumorais Cultivadas
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