RESUMO
AIM: In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells. MATERIALS AND METHODS: EAT cells were exposed to various AsA (0-10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry. RESULTS: Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10-24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6-24 h after treatment. CONCLUSION: These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exerted by AsA alone.
Assuntos
Antineoplásicos/farmacologia , Ácido Ascórbico/farmacologia , Carcinoma de Ehrlich/terapia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Terapia Combinada , Ensaios de Seleção de Medicamentos Antitumorais , Hipertermia Induzida , Espécies Reativas de Oxigênio/metabolismoRESUMO
The evaluation of iron status in dialysis patients provides information essential to the planning of adequate recombinant human erythropoietin treatment. The cellular iron status of the patients can be determined from the recently available measurement of reticulocyte hemoglobin equivalent (RET-He). RET-He is measured on the basis of automated fluorescent flow cytometry which in the reticulocyte channel, using a polymethine dye, also measures the mean value of the forward light scatter intensity of mature red blood cells and reticulocytes. These values equate with reticulocyte hemoglobin content. In this study, to clarify the accuracy of RET-He in diagnosing iron deficiency in dialysis patients, we initially compared RET-He with such iron parameters as serum ferritin levels, transferrin saturation and content of reticulocyte hemoglobin (CHr) which has been established as indicators of functional iron deficiency. Secondly, we investigated the changes in RET-He during iron supplementation for iron-deficient patients to determine whether this marker is a prospective and reliable indicator of iron sufficiency. The participants in this study were 217 haemodialysis patients. Iron deficiency was defined as havsing a transferrin saturation (TSAT) < 20% or serum ferritin < 100 ng/ml. Conventional parameters of red blood cells and RET-He were measured by on a XE-2100 automated blood cell counter (Sysmex). CHr was measured on an ADVIA120 autoanalyser (Siemens). RET-He mean value was 32.4 pg and good correlation (r = 0.858) between RET-He and CHr is obtained in dialysis patients. Receiver operating characteristic curve analysis revealed, values of the area was 0.776 and at a cutoff value of 33.0 pg, a sensitivity of 74.3% and a specificity of 64.9%, were achieved. Iron supplements given to the patients with low TSAT or ferritin, RET-He responded within 2 weeks, and this seemed to be a potential advantage of using RET-He in the estimation of iron status. RET-He is a new parameter, equivalent value to CHr, and is easily measurable on the widely spread and popular blood cell counter and is a sensitive and specific marker of iron status in dialysis patients.
Assuntos
Anemia Ferropriva/diagnóstico , Hemoglobinas , Deficiências de Ferro , Ferro/sangue , Diálise Renal , Reticulócitos/química , Biomarcadores/química , Humanos , Curva ROC , Contagem de ReticulócitosRESUMO
AIM: Hydrogen-dissolved water (HD-water) or platinum nanocolloid (Pt-nc) has been individually expected as a new therapeutic agent for oxidative stress-related diseases, whereas little is known about their combined effects on cancer, which were elucidated in the present study. METHODS: HD-water was prepared by microporous gas bubbling, and supplemented with Pt-nc consisting of 0.003-1 ppm Pt and PVP polymers. Antioxidant activities were examined by 1, 1-diphenyl-picrylhydrazyl (DPPH)-radicalscavenging assay. Cytotoxic activities were examined by culturing of tumor and normal cell lines, respectively. RESULTS: HD-water accelerated the Pt-nc-based DPPH-radical scavenging. Pt-nc-supplemented HD-water inhibited either colony formation efficiencies or colony sizes of human tongue carcinoma cells HSC-4, in contrast to no effects of HD-water alone, Pt-nc alone or Pt-absent PVP, but not appreciably inhibit normal human tongue epithelial-like cells DOK. Pt-nc-supplemented HD-water also suppressed cell population growth of HSC-4 cells of near-confluence (at higher cell densities) in view of decreases in either cell numbers or mitochondrial function, although less markedly than colony formation starting from a sparse-cell state (at lower cell densities). Dissolved hydrogen, oxygen concentration or oxido-reduced potentials of HD-water was decreased, rather decreased or increased by Pt-nc addition, respectively. CONCLUSIONS: Anti-cancer activity of Pt-nc-supplemented HD-water was shown by its preferential cell-growth inhibition to human tongue carcinoma cells HSC-4 over normal human tongue cells DOK, and might be partly attributed to HD-water-caused enhancement of Pt-nc-relevant antioxidant ability. Pt-nc-supplemented HD-water is expected as a novel agent against human tongue cancers due to its cancer progression-repressive abilities.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Hidrogênio/química , Platina/química , Neoplasias da Língua/prevenção & controle , Água/farmacologia , Compostos de Bifenilo , Sobrevivência Celular/efeitos dos fármacos , Coloides/química , Ensaio de Unidades Formadoras de Colônias , Sequestradores de Radicais Livres , Humanos , Nanopartículas Metálicas , Picratos , Neoplasias da Língua/patologia , Água/químicaRESUMO
AIM: To evaluate promotive effects of hyperthermia on antitumor activity of new delta-alkyllactones (DALs) of low molecular weight (184-254 Da), chemically synthesized, which are different from natural macrocyclic lactones of high molecular weight (348-439 Da), such as camptothecin and sultriecin. METHODS: A suspension of Ehrlich ascites tumor (EAT) cells was mixed with a DAL in a glass tube, heated at 37 or 42 degrees C for 30 min in a water bath, and cultured at 37 degrees C for 20 or 72 h. Cell viability was measured by the mitochondrial dehydrogenase- based WST-1 assay. DALs incorporated into EAT cells was extracted and measured by gas-liquid chromatography. RESULTS: The reduction of cell viability by DALs was markedly enhanced upon the treatment at 42 degrees C compared to that at 37 degrees C. At 37 degrees C, delta-hexadecalactone (DH16:0) and delta-tetradecalactone (DTe14:0) displayed cytostatic activity (at 100 microM survival level: 20.7%, 66.1%; at 50 microM--41.2%, 82.4%, respectively). Their activity was more marked at 42 degrees C (at 100 microM 10.6%, 27.6%; at 50 microM 30.6, 37.5%, ibid). The other DALs, delta-undecalactone (DU11:0), delta-dodecalactone (DD12:0), and delta-tridecanolactone (DTr13:0) were almost ineffective. Evaluation of survival rate in the cells treated for 30 min by DALs with the next culturing of EAT cells for 72 h resulted in the enhanced carcinostatic activity of DH16:0 and DTe14:0 even at concentrations as low as 25 microM at either 37 degrees C (18.5%, 78.5%, ibid) or 42 degrees C (5.0%, 42.0%, ibid), but the others exhibited slight activity or none. DH16:0 was effective at either 37 degrees C (36.0%) or 42 degrees C (23.0%) even at a lower dose of 10 microM. At the same time only the most cytostatic DH16:0 was incorporated into EAT cells and the rate of incorporation was more at 42 degrees C than at 37 degrees C. CONCLUSION: Delta-hexadecanolactone (DH16:0) exhibited the most cytostatic effect that was significantly enhanced by hyperthermia. It allows to consider it as a potent antitumor agent, especially in combination with hyperthermia.
Assuntos
Carcinoma de Ehrlich/terapia , Hipertermia Induzida , Lactonas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Químicos , Oxirredução , TemperaturaRESUMO
AIM: To evaluate promotive effect of hyperthermia on the carcinostatic activity of synthesized omega-hydroxy fatty acids (omega HFAs) and their ethylesters agaist Ehrlich ascites tumor (EAT) cells. METHODS: EAT cells were cultured with either omegaHFAs or their ethylester derivatives in a water bath at either 37 degrees C or 42 degrees C for 30 min, followed by incubation in a CO2 incubator for 20 or 72 h. Mitochond-rial dehydrogenase-based WST-1 assay and trypan blue dye exclusion assay were then conducted after incubation. Morphological changes were observed by scanning electron microscopy (SEM). RESULTS: Omega-HFA having a saturated 16-carbon straight-chain (omega H16:0) was the most carcinostatic (at 37 degrees C - viability level: 60.0%; at 42 degrees C - 49.6% (WST-1)) among saturated and unsaturated omegaHFAs with 12, 15 or 16 carbon atoms, when administrated to EAT cells at 100 microM for 20 h. Carcinostatic activity was markedly enhanced by ethyl-esterization of saturated fatty acids, such as omegaH16:0 (at 37 degrees C - 42.3%; at 42 degrees C - 11.2%, ibid) and omegaH15:0 (at 37 degrees C - 74.6%; at 42 degrees C - 25.3%, ibid), and their unsaturated counterparts were extremely effective only in combination with hyperthermia. Prolongation of the incubation period to 72 h at the same concentration increased appreciably their carcinostatic effect (omega H16:0 ethylesther: 1.3%; omegaH15:0 ethylesther: 8.0%). These values were also supported by dye exclusion assay. The carcinostatic activity enhanced more markedly by hyperthermia (1.2%; 2.1%, ibid). SEM shows that omegaH16:0 ethylester-exposed EAT cells underwent extensive injury, such as deformation of cell structure or disappearance of microvilli. CONCLUSIONS: omega H16:0 ethylester possesses high carcinostatic activity in vitro in combination with hyperthermia and may be utilized as potent anticancer therapeutic agent.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Carcinoma de Ehrlich/tratamento farmacológico , Ácidos Graxos/metabolismo , Ácidos Graxos/uso terapêutico , Temperatura Alta , Animais , Antineoplásicos/química , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/ultraestrutura , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Esterificação , Ácidos Graxos/química , Feminino , Camundongos , Camundongos Endogâmicos ICR , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Fatores de Tempo , Células Tumorais Cultivadas/metabolismoRESUMO
AIM: To evaluate inhibitory effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on DNA synthesis in combination with hyperthermia in vitro. METHODS: A suspension of Ehrlich ascites tumor cells (EAT) was mixed with DHA or EPA in a glass tube, heated at 37 degrees C, 40 degrees C, or 42 degrees C for 1 h in a water bath, and cultured at 37 degrees C for 19 or 96 h. DNA synthesis was assayed by monitoring of the incorporation of [3H]-thymidine into the acid-insoluble fraction. DHA or EPA incorporated into EAT cells was extracted and measured by thin-layer chromatography and gas-liquid chromatography. RESULTS: The inhibition of DNA synthesis by EPA or DHA increased markedly upon the treatment at 42 degrees C and 40 degrees C compared to that at 37 degrees C. At 37 degrees C, inhibitory action of EPA was more potent than that of DHA at low concentrations (at 50 microM -- DNA synthesis level: EPA, 63.1%; DHA, 87.9%), whereas inhibitory action of DHA was higher at 150 muM (16.7%, 4.4%, ibid.). The effect of DHA compared to EPA was more marked at 40 degrees C (29.0%, 19.2% at 100 microM) or 42 degrees C (19.7%, 10.6% at 100 microM). Evaluation of DNA synthesis rate in the cells treated for 1 h by EPA or DHA with the next culturing of EAT cells for 19 h resulted in the enhanced inhibitory activity of EPA even at concentrations as low as 50 microM at either 37 degrees C (0.5%, 11.3%) or 42 degrees C (0.6%, 4.5%), which in these conditions was higher than that of DHA. At the same time the rate of incorporation of EPA in EAT cells at 37 degrees C or 42 degrees C was lower than that of DHA. CONCLUSION: Administration of DHA or EPA in vitro significantly inhibit DNA synthesis, and such effect is enhanced by combination of PUFAs with hyperthermia.
Assuntos
Carcinoma de Ehrlich/genética , Replicação do DNA/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Temperatura Alta , Animais , Transporte Biológico , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Células Tumorais CultivadasRESUMO
To elucidate the molecular mechanism of colorectal carcinogenesis, we have been attempting to isolate genes involved in the beta-catenin/T-cell factor pathway. In the experiments reported here, analysis by cDNA microarray indicated that AF17, a fusion partner of the MLL gene in acute leukemias with t(11;17)(q23;q21), was transactivated according to accumulation of beta-catenin. Expression of AF17 was significantly enhanced in 8 of the 12 colorectal cancer tissues examined. Introduction of a plasmid designed to express AF17 stimulated growth of NIH3T3 cells, and fluorescence-activated cell sorter analysis indicated that the AF17 regulation of cell-cycle progression was occurring mainly at the G(2)-M transition. Our results suggest that the AF17 gene product is likely to be involved in the beta-catenin-T-cell factor/lymphoid enhancer factor signaling pathway and to function as a growth-promoting, oncogenic protein. These findings should aid development of new strategies for diagnosis, treatment, and prevention of colon cancers and acute leukemias by clarifying the pathogenesis of these conditions.
Assuntos
Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias/genética , Transativadores , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Células COS , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Chlorocebus aethiops , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/fisiologia , Transcrição Gênica , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , beta CateninaRESUMO
The carcinostatic activity has been studied for fatty acids of diverse species but scarcely for fatty alcohols. Three unsaturated fatty alcohols at 35-50 microM inhibited DNA synthesis and the proliferation of tumor cells by a combination with hyperthermia to greater extents in the order: oleyl (C18:1)-> linoleyl (C18:2)-> alpha-linolenyl (C18:3) alcohol, which is an order inverse to that known for the corresponding fatty acids (4). In contrast, two saturated fatty alcohols, palmityl (C16:0)- and stearyl (C18:0) alcohols, did not inhibit at the same concentrations. At 100 microM, palmityl alcohol inhibited, whereas stearyl alcohol did not. The effective fatty alcohols appreciably permeated the cells. The inhibition of the unsaturated fatty alcohols on DNA synthesis and proliferation was nearly proportional to the amount of their intercellular accumulation at 37 degrees C or 42 degrees C; the most inhibitory, oleyl alcohol, was the most membrane-permeable, whilst inversely the least inhibitory, alpha-linolenyl alcohol, was the least permeable. A proportional correlation was not observed for saturated fatty alcohols; palmityl alcohol underwent an approximate 2-fold more abundant accumulation than other fatty alcohols, but was weakly inhibitory. Thus, oleyl alcohol may exert an antitumor action via appropriately efficient transmembrane permeation and a combination with hyperthermia.
Assuntos
Carcinoma de Ehrlich/tratamento farmacológico , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Álcoois Graxos/farmacologia , Temperatura Alta , Animais , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patologia , Divisão Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultura , Álcoois Graxos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICR , Células Tumorais Cultivadas/metabolismoRESUMO
Accumulation of beta-catenin in cytoplasm and nuclei is frequently observed in a wide variety of tumors arising, for example, in the colon, liver, uterus, or brain. In association with Tcf/LEF transcription factors, beta-catenin regulates expression of genes involved in the Wnt/wingless signaling pathway, but the precise mechanisms are unclear. Here we report evidence that the claudin-1 (CLDNI) gene is one of the genes regulated by beta-catenin. Not only did expression of CLDN1 decrease significantly in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of wild-type APC into the APC-deficient colon cancer cells, but also two putative Tcf4 binding elements in the 5' flanking region of CLDN1 were confirmed to be responsible for activating its transcription. We documented increased expression of CLDN1 in all 16 primary colorectal cancers we examined, compared with adjacent noncancerous mucosae. Furthermore, immunohistochemical staining demonstrated that claudin-1 was weakly stained at apical boarder of lateral membrane of noncancerous epithelial cells and that it was strongly stained at all cell-cell boundaries and in the cytoplasms of cancer cells. Our results imply that claudin-1 is involved in the beta-catenin-Tcf/LEF signaling pathway, and that increased expression of claudin-1 may have some role in colorectal tumorigenesis.
Assuntos
Neoplasias do Colo/fisiopatologia , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/fisiologia , Neoplasias Retais/fisiopatologia , Transdução de Sinais/fisiologia , Junções Íntimas/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Claudina-1 , Colo/citologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/patologia , Primers do DNA , Genes Reporter , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Proteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Transdução de Sinais/genética , Junções Íntimas/genética , Células Tumorais Cultivadas , beta CateninaRESUMO
Hemodialysis (HD) patients have accelerated atherosclerosis. Recent reports have shown that aortosclerosis is more frequently observed in HD patients than in healthy subjects. Macrophage colony-stimulating factor (M-CSF) secreted by activated macrophages may be involved in the process of aortosclerosis in HD patients. To understand the mechanism behind the increased incidence of aortosclerosis in HD patients, we examined the relationships between serum M-CSF levels and aortic calcification index (ACI) estimated by CT scan. A significant increase in serum M-CSF concentrations was found in HD patients (3.8 +/- 0.2 ng/ml) as compared with controls (1.5 +/- 0.1 ng/ml). No significant differences were observed between chronic glomerulonephritis and diabetes mellitus groups of patients. We also found no significant differences between the groups using different membranes (triacetate 3.8 +/- 0.2 ng/ml vs. polysulfone 3.8 +/- 0.4 ng/ml). There was no correlation between serum M-CSF concentrations and clinical parameters such as age, duration of HD, blood pressure, serum concentrations of nitrogen, creatinine, cholesterol, triglyceride, LDL, Ca x P products, and intact parathyroid hormone. A positive correlation was observed between serum M-CSF levels and ACI in HD patients (r = 0.596, p < 0.01). These results suggest that M-CSF may be involved in the process of aortosclerosis in HD patients.
Assuntos
Doenças da Aorta/diagnóstico por imagem , Calcinose/diagnóstico por imagem , Fator Estimulador de Colônias de Macrófagos/sangue , Diálise Renal , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estatísticas não Paramétricas , Tomografia Computadorizada por Raios XRESUMO
Tumor metastasis and invasion were shown to be inhibited by the 2-O-phosphorylated form (Asc2P) of L-ascorbic acid (Asc); intact Asc did not inhibit tumor invasion when added once, but appreciably inhibited it upon repeated addition. The anti-metastatic effect is attributable to a marked enrichment of intracellular Asc by Asc2P, subsequently dephosphorylated. Asc2P scavenged most of the intracellular reactive oxygen species (ROSin), and notably inhibited production of matrix metalloproteases and cell motility. ROSin was decreased by Asc2P more markedly than by Asc added once. Thus, involvement of ROSin in tumor invasion and a potent anti-metastatic therapy by ROSin-decreasing agents are suggested.
Assuntos
Antineoplásicos/uso terapêutico , Ácido Ascórbico/metabolismo , Ácido Ascórbico/uso terapêutico , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Animais , Antineoplásicos/farmacocinética , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacocinética , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/prevenção & controle , Fibrossarcoma/secundário , Sequestradores de Radicais Livres , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/prevenção & controle , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
Beta-catenin plays significant roles in cell-to-cell adhesion and the Wnt/Wg signal transduction pathway. Accumulation of this protein in the cytoplasm and nucleus as a result of mutations of the adenomatous polyposis coli tumor suppressor gene or of the beta-catenin gene itself is often seen in a wide variety of tumors including carcinomas of the colon, liver, uterus, and brain. Interaction of accumulated beta-catenin with Tcf/Lef transcription factors is known to deregulate expression of some downstream genes, but the precise mechanisms whereby beta-catenin contributes to carcinogenesis remain to be disclosed. Here we report isolation of a novel murine gene, Drctnnb1a (down-regulated by Ctnnb1, a), the expression of which was experimentally down-regulated in response to the activated form of beta-catenin. To investigate a possible role of DRCTNNB1A in cancers, we also isolated the human homologue, DRCTNNB1A, the deduced product of which was 91% identical to the murine protein. The transcript was expressed in all human tissues examined, and we assigned the genomic location of DRCTNNB1A to chromosomal band 7p15.3 by in situ hybridization. Expression of DRCTNNB1A in SW480 colon cancer cells was significantly increased in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of the beta-catenin-binding domain of the adenomatous polyposis coli gene into the cells. Furthermore, we documented reduced expression of DRCTNNB1A in 12 of 15 primary colorectal cancers examined, compared with corresponding adjacent noncancerous mucosae. Our results implied that DRCTNNB1A is one of the genes involved in the beta-catenin-Tcf/Lef signaling pathway, and that reduced expression of DRCTNNB1A may have some role in colorectal carcinogenesis.
Assuntos
Neoplasias do Colo/genética , Proteínas do Citoesqueleto/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/genética , Transativadores , Transcrição Gênica , Animais , Células COS , Caderinas/genética , Caderinas/fisiologia , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células L , Proteínas de Membrana , Camundongos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , beta CateninaRESUMO
beta-Catenin, a key regulator of cellular proliferation, is often mutated in various types of human cancer. To investigate cellular responses related to the beta-catenin signaling pathway, we applied a differential display method using mouse cells transfected with an activated form of mutant beta-catenin. This analysis and subsequent northern-blot hybridization confirmed that expression of a murine gene encoding NBL4 (novel band 4.1-like protein 4) was up-regulated by activation of beta-catenin. To examine a possible role of NBL4 in cancer, we isolated the human homologue of the murine NBL4 gene by matching mNBL4 against the human EST (expressed sequence tag) database followed by 5' rapid amplification of cDNA ends (5'RACE). The cDNA of hNBL4 encoded a protein of 598 amino acids that shared 87% identity in amino acid sequence with murine NBL4 and 71% with zebrafish NBL4. A 2.2-kb hNBL4 transcript was expressed in all human tissues examined with high levels of expression in brain, liver, thymus and peripheral blood leukocytes and low levels of expression in heart, kidney, testis and colon. We determined its chromosomal localization at 5q22 by fluorescence in situ hybridization. Expression of hNBL4 was significantly reduced when beta-catenin was depleted in SW480 cells, a human cancer cell line that constitutionally accumulates beta-catenin. The results support the view that NBL4 is an important component of the beta-catenin / Tcf pathway and is probably related to determination of cell polarity or proliferation.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/genética , Transdução de Sinais/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Northern Blotting , Cromossomos Humanos Par 5 , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Células L , Fator 1 de Ligação ao Facilitador Linfoide , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Ativação Transcricional , Transfecção , Peixe-Zebra , beta CateninaRESUMO
We have previously shown that p26olf is a novel S100-like Ca(2+)-binding protein in the frog olfactory epithelium. In this paper, we characterized the Ca(2+) binding property of p26olf, examined the precise localization in the frog olfactory epithelium, and searched for the possible target proteins of p26olf. By flow dialysis experiments using (45)Ca, p26olf was suggested to bind approximately 4 Ca(2+). Circular dichroism measurement showed that binding of Ca(2+) to p26olf induces an increase in the apparent content of both alpha-helix and beta-sheet with an apparent K(d) value of 2.4 micrometer. Electron microscopic observation disclosed p26olf immunoreactivity in the cilia, dendritic knob, and dendrite of olfactory receptor cells. Blot overlay analysis and affinity purification of p26olf-binding proteins showed that p26olf binds to a frog beta-adrenergic receptor kinase-like protein in a Ca(2+)-dependent manner. These results suggested that p26olf has some roles in the olfactory transduction or adaptation.
Assuntos
Proteínas de Anfíbios , Proteínas de Ligação ao Cálcio/metabolismo , Mucosa Olfatória/metabolismo , Proteínas S100/metabolismo , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imuno-Histoquímica , Ligação Proteica , Conformação Proteica , Rana catesbeiana , Proteínas S100/química , Quinases de Receptores Adrenérgicos betaRESUMO
The invasion of human fibrosarcoma HT-1080 cells through Matrigel was shown to be inhibited by pretreatment with ascorbic acid (Asc) or its four derivatives, such as Asc-6-O-palmitate (Asc6Plm), Asc-2-O-phosphate (Asc2P), Asc-2-O-phosphate-6-O-palmitate (Asc2P6Plm), and Asc-5,6-benzylidene (Asc5,6Bz) of non-cytotoxic concentrations for 1 or 18 hr. Two lipophilic derivatives such as Asc6Plm and Asc2P6Plm exerted an invasiveness-inhibitory activity more markedly with 1-hr pretreatment, being a more practical index in terms of the plasma half-life, than Asc, Asc5,6Bz or Asc2P being less lipophilic. Considerably less cytotoxicity (a > 3.3-fold higher IC50 for 1-hr pretreatment) of Asc2P6Plm sufficiently compensated a slightly lower invasiveness-inhibitory activity (a < 1.8-fold higher EC50) as compared with Asc6Plm. Pulmonary metastasis of mouse melanoma B16BL6 cells injected into the tail vein was also inhibited by intravenous administration with Asc2P6Plm dose-dependently. Thus Asc2P6Plm, a lipophilicity-hydrophilicity balanced molecule protectively blockd in the autooxidation-prone moiety, is anticipated as a potent anti-metastatic agent via inhibition of tumor invasion.
Assuntos
Antineoplásicos/uso terapêutico , Ácido Ascórbico/análogos & derivados , Compostos de Benzilideno/uso terapêutico , Fibrossarcoma/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/secundário , Invasividade Neoplásica/prevenção & controle , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ácido Ascórbico/química , Ácido Ascórbico/farmacologia , Ácido Ascórbico/uso terapêutico , Compostos de Benzilideno/química , Compostos de Benzilideno/farmacologia , Sobrevivência Celular , Fenômenos Químicos , Físico-Química , Colágeno , Combinação de Medicamentos , Feminino , Meia-Vida , Laminina , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Proteoglicanas , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/transplanteRESUMO
The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.
Assuntos
Carcinoma Hepatocelular/metabolismo , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas/fisiologia , Proteínas Repressoras , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Peixe-Zebra , Proteína da Polipose Adenomatosa do Colo , Adenoviridae/genética , Apoptose/genética , Proteína Axina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Carcinoma Hepatocelular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Análise Mutacional de DNA , Genes APC , Predisposição Genética para Doença , Vetores Genéticos/genética , Quinase 3 da Glicogênio Sintase , Humanos , Neoplasias Hepáticas/genética , Substâncias Macromoleculares , Proteínas de Neoplasias/genética , Polimorfismo Conformacional de Fita Simples , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas Wnt , beta CateninaRESUMO
Invasion of rat fibroblastic cells Rat-1 through Matrigel was shown to be promoted by transfection with tax gene of human T-cell leukemia virus type 1. We found that an oxidation-resistant type of vitamin C (Asc), Asc-2-O-phosphate (Asc2P), inhibited the invasion of the tax-transfected Rat-1 cells (W4 cells). Intracellular Asc (Ascin), after enzymatic dephosphorylation of administered Asc2P, was more abundant in W4 cells than in Rat-1 cells, and the ratio of dehydroascorbic acid versus Asc was increased in W4 cells but scarcely in Rat-1 cells, according to the enhanced level of intracellular reactive oxygen species (ROSin) in W4 cells. Asc2P notably repressed the increases in both ROSin and secretion of matrix metalloproteases (MMPs), but did not affect Tax protein expression in tax-transfectants. NF-kappa B activation, as evidenced by its translocation to the nucleus in W4 cells, was also repressed by Asc2P. Thus, the tax-promoted invasion together with the enhanced production of MMPs occurred with NF-kappa B activation and the increase in ROSin, both of which were effectively reduced by Asc2P. These findings indicate the therapeutic efficacy of Ascin-enriching agents for the prevention against tumor invasion in which ROSin plays a major role.
Assuntos
Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Produtos do Gene tax/genética , NF-kappa B/metabolismo , Animais , Membrana Basal/citologia , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Fibroblastos/citologia , Genes pX , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Ratos , TransfecçãoRESUMO
Docosahexaenoic acid (DHA) inhibited the DNA synthesis in Ehrlich ascites tumor cells more markedly than eicosapentaenoic acid (EPA), which was more inhibitory than oleic-, linoleic-, linolenic-, and arachidonic acids. Their activities augmented according to the increase of number of double bonds in the molecule. To correlate the cytotoxicity with lipid syntheses in the cells, distribution of EPA and DHA incorporated into cellular lipids was assessed. EPA was incorporated into triglycerides (TG) and DHA into phosphatidylcholine (PC). These synthesis into TG and PC etc., which shattered from cytotoxicity, may be involved in tumor-cellular protecting actions against EPA or DHA. EPA and DHA involved in cytotoxicity exhibition are their free acid forms. Thus, as an anticancer reaction, intracellular accumulation in the free acid form of DHA was more marked than that of EPA.
Assuntos
Antineoplásicos/farmacologia , Proteínas Sanguíneas/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Metabolismo dos Lipídeos , Animais , Carcinoma de Ehrlich/tratamento farmacológico , DNA/biossíntese , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Hepatic ischemia was induced by clamping the hepatic artery, portal vein, and bile duct. After 15 min of ischemia, the hepatic glutathione (GSH) content rapidly decreased. On the other hand, after the start of reperfusion, the hepatic GSH levels promptly increased and reached a peak at about 1 h, and thereafter decreased to a minimum level by 2 h. Under such conditions, we examined the changes in the methionine adenosyltransferase (MAT) activity in the liver. Though the time course of MAT activity was somewhat delayed compared with that of the hepatic GSH levels, both patterns were substantially similar during ischemia-reperfusion. In contrast to the changes in the MAT activity during ischemia-reperfusion, the levels of MAT protein were unchanged during these periods. When endogenous antioxidant coenzyme Q(10) (CoQ(10)) was administered to rats prior to ischemia, both the reduction in the MAT activity and hepatic GSH levels induced by ischemia-reperfusion were protected. Our findings suggest that CoQ(10) may posttranslationally regulate the MAT activity via the changes in the GSH level in the liver.
Assuntos
Glutationa/metabolismo , Fígado/metabolismo , Metionina Adenosiltransferase/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Antioxidantes/farmacologia , Coenzimas , Citoproteção , Fígado/irrigação sanguínea , Masculino , Ratos , Ratos Wistar , Fatores de Tempo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Conventional studies on vitamin C have been conducted by single-dosage administration with ascorbic acid itself being so labile as to undergo irreversible degradation. In contrast, enrichment of intracellular ascorbate is accomplished by pro-vitamin C in which its 2,3-enediol moiety is protected with phosphate ester, being thereafter enzymatically esterolyzed. Ascorbic acid-2-O-phosphate (Asc2P) gradually releases ascorbate, which is cumulatively taken up into diverse human or mammalian cells, and prevents cell injuries such as post-ischemic reperfusional injury in the liver or heart, age-dependent telomeric DNA shortening in endothelial or epithelial cells, UV-B irradiational injury in the skin and lipid peroxide-induced injury in the vascular endothelium, and tumor invasion and metastasis.