RESUMEN
Transcription factor decoy oligodeoxynucleotides (ODN) represent a novel tool for targeted inhibition of the STAT3 signaling pathway. To investigate its therapeutic potential in ovarian cancer, a double-stranded decoy ODN mimicking STAT3-specific cis-elements was transfected into two ovarian cancer cell lines OVCAR3 and SKOV3. The STAT3 decoy ODN treatment specifically blocked STAT3 signaling, and inhibited cell proliferation by inducing apoptosis and cell cycle arrest. These results suggest that targeted blockade of the STAT3 signaling pathway with a decoy ODN may represent a potential therapeutic approach in the treatment of ovarian cancer.
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Proliferación Celular , Oligodesoxirribonucleótidos/metabolismo , Neoplasias Ováricas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Oligodesoxirribonucleótidos/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Factor de Transcripción STAT3/genética , Factores de Tiempo , TransfecciónRESUMEN
Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45âmmol/l for A-ESCs and 7.87âmmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17ß-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17ß-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater effect of metformin on A-ESCs from secretory phase.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenomiosis/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Endometrio/efectos de los fármacos , Metformina/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Células del Estroma/efectos de los fármacos , Adenomiosis/metabolismo , Adenomiosis/patología , Adulto , Apoptosis/efectos de los fármacos , Western Blotting , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Endometrio/metabolismo , Endometrio/patología , Femenino , Humanos , Hipoglucemiantes/farmacología , Técnicas para Inmunoenzimas , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
BACKGROUND/AIMS: although numerous studies have explored the mechanisms regulating the enzyme activity of NADPH oxidase in diabetic nephropathy (DN), little information is available for the contribution of microRNAs (miRNAs) to the regulation of NADPH oxidase expression. Therefore, the present study was to test whether miRNAs importantly contribute to the regulation of NOX4 expression, a major catalytic subunit of NADPH oxidase under hyperglycemia. METHODS: diabetic rats were induced by streptozotocin. miRNA microarray, Western blot, real-time RT-PCR and luciferase reporter assays were employed in this study. RESULTS: among 5 miRNAs, which are predicted to have a binding capacity to rat NOX4, the miRNA-25 level was significantly reduced both in the kidney from diabetic rats and in high glucose-treated mesangial cells, accompanied by the increases in NOX4 expression levels. In an in vitrostudy, we found that NADPH activity was increased by 226.2% in miRNA-25 inhibitor transfected cells and decreased by 51.0% in miRNA-25 precursor transfected cells. miR-25 inhibitor dramatically increased both NOX4 mRNA and protein levels. We then showed that miR-25 negatively regulated NOX4 expression by directly targeting the 3'-UTR by luciferase reporter assays. It was found that transfection of miR-25 precursor significantly decreased the luciferase activity of NOX4 3'-UTR by 39.5%, whereas the mutant sequence restored levels to 79.4%. Finally, our results indicated that the miR-25-mediated NOX4 mRNA level may result from the regulation of mRNA stability. CONCLUSIONS: these findings for the first time indicate that miRNA-25 may serve as an endogenous gene silencing factor and contributes to the regulation of NOX4 expression and function in DN.
Asunto(s)
Nefropatías Diabéticas/enzimología , MicroARNs/metabolismo , NADPH Oxidasas/genética , Animales , Células Cultivadas , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/genética , Expresión Génica , Silenciador del Gen , Masculino , Células Mesangiales/enzimología , Células Mesangiales/metabolismo , MicroARNs/genética , Modelos Animales , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
OBJECTIVE: To explore the stroma-epithelium interactions in endometriosis and to identify the possible signalling pathways involved in this cross-talk. DESIGN: Laboratory study via primary cultured endometrial stromal and epithelial cells. SETTING: University Hospital. PATIENTS: Fifteen patients with endometriosis confirmed by histopathology were recruited in the study, and 12 women free of endometriosis were used as control group. INTERVENTION(S): Specific NFkappaB inhibitor 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) was used in cell cultures. MAIN OUTCOME MEASURE(S): The expression and secretion of MMP-2, MMP-9, TIMP-1, TIMP-2 and the DNA-binding activity of NFkappaB in normal endometrial stromal cells or in co-cultures with normal or endometriotic epithelial cells from patients with endometriosis. RESULT(S): Endometrial epithelial cells induced MMP-9 and MMP-2 expression in normal stromal cells in vitro. In co-cultures with endometriotic epithelial cells, normal endometrial stromal cells expressed and secreted higher MMP-2 (p < 0.05) and MMP-9 (p < 0.05). Specific inhibition of NFkappaB pathway in stromal cells abolished this induction effect by epithelial cells. CONCLUSION(S): Endometriotic epithelial cells induce MMPs expression and secretion in normal endometrial stromal cells via an NFkappaB-dependent pathway in vitro. This cross-talk between epithelial cells and stromal cells may facilitate the implantation and extension of the ectopic foci and favour the development of the disease.
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Endometriosis/metabolismo , Endometrio/metabolismo , Células Epiteliales/metabolismo , Metaloproteinasas de la Matriz/biosíntesis , FN-kappa B/metabolismo , Células del Estroma/metabolismo , Adulto , Endometriosis/fisiopatología , Femenino , Humanos , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Persona de Mediana Edad , Transducción de SeñalRESUMEN
In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.
Asunto(s)
Endometriosis/metabolismo , Endometriosis/patología , Endometrio/citología , Endometrio/metabolismo , Células Epiteliales/metabolismo , Células del Estroma/metabolismo , Adulto , Western Blotting , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Endometrio/patología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/fisiología , Femenino , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/metabolismo , Morfolinas/farmacología , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/fisiología , SurvivinRESUMEN
Although radiotherapy is routinely administered to high-risk endometrial carcinoma and offer a significant disease-free survival advantage, the therapeutic effect is sometimes limited by the occurrence of radioresistance. To determine the patterns of gene expression responsible for the radioresistance and to search for potential target genes for radiotherapy, we selected two cell lines with distinct radiosensitivities using colony-formation assay from four endometrial cancer cell lines. The cell cycle distribution showed higher fractions of G2/M phase cells in the radiosensitive cell line KLE after radiation compared with the radioresistant cell line ISK. Apoptosis assessment also showed significant elevation in the percentage of early apoptosis cells in KLE cells. Subsequently, gene expression changes after X-ray exposure were analyzed by using oligonucleotide microarrays. We identified, respectively, in ISK and KLE, 227 and 354 genes that exhibited > or =2-fold difference. However, only 53 genes showing differences more than double the median expression value between the two groups were defined as radiosensitivity (or radioresistance)-related genes. Among these, genes associated with DNA-repair, apoptosis, growth factor, signal transduction, cell cycle and cell adhesion were predominant. The validity of the expression level of 10 randomly selected genes was confirmed by real-time PCR and/or Western blotting. In conclusion, the differential gene expression changes that occur after radiation in the two cell lines will provide insight into molecular mechanisms of radioresistance in endometrial carcinoma, and also the means to find potential targets to achieve further gains in therapeutic benefit.
Asunto(s)
Neoplasias Endometriales/genética , Expresión Génica/efectos de la radiación , Tolerancia a Radiación/genética , Apoptosis/efectos de la radiación , Western Blotting , Línea Celular Tumoral , Neoplasias Endometriales/radioterapia , Femenino , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Radiación Ionizante , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
It is well-known that exposure to unopposed estrogen is considered as an important risk factor for endometrial cancer. Recent studies have shown that over-expression of DNA methyltransferases (DNMTs) are involved in the development of endometrial cancer. Therefore, the present study was undertaken to elucidate the impact of estrogen on the expression of DNMTs in endometrial cancer. Ishikawa cell line was used. Flow cytometry analysis demonstrated that 17 beta-estradiol (E(2)) enhanced the cell proliferation with a peak at 10(-8) M. Over-expression of DNMT3B treated with E(2) was confirmed by real-time PCR and western blotting analysis. Furthermore, the up-regulation of DNMT3B expression induced by E(2) was suppressed by the addition of ICI182780. However, we did not observe changes in the expression of DNMT1. Our study suggests that estrogen up-regulating the expression of DNMT3B in an ER-dependent pathway may be a possible mechanism for estrogen facilitates the malignant transformation of endometrial cancer cells.
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Adenocarcinoma/enzimología , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Neoplasias Endometriales/enzimología , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Adenocarcinoma/genética , Adenocarcinoma/patología , Análisis de Varianza , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ADN Metiltransferasa 3BRESUMEN
AIM: To investigate the role of heat shock proteins 70 (HSP70) in radiosensitivity and invasiveness of endometrial cancer in vitro. METHODS: HSP70 expression was silenced in relatively radioresistant, well-differentiated human endometrial cancer cell line ISK, using small interference RNA method, or by HSP70 overexpression after transfecting a HSP70-expressing vector. The effect of HSP70 on ISK cell line response to irradiation was evaluated. The surviving fraction was measured using colony-formation assay. Apoptosis was detected by flow cytometry and HSP70 expression was determined by quantitative real-time polymerase chain reaction, western-blot, and/or immunocytochemistry. Cell invasiveness was measured using transwell invasion assay. RESULTS: HSP70 silencing caused a significant increase in irradiation-induced cell killing in comparison with control cells, with an enhancement factor of 1.27, and in the percentage of apoptotic cells (14.22% vs 6.74%, P = 0.021). After 4 Gy irradiation, mean +/- standard deviation survival fraction in ISK cells was reduced to 0.32 +/- 0.04 in comparison with control values but in ISK/siRNA-HSP70 cells the survival fraction was higher and amounted to 0.51 +/- 0.08 (P = 0.026). Silencing HSP70 significantly inhibited cell invasion before and after irradiation (106 +/- 19 vs 219 +/- 18 and 119 +/- 16 vs 256 +/- 31, P = 0.007). On the contrary, ectopic overexpression of HSP70 attenuated irradiation-induced apoptosis (7.15% vs 4.08%, P = 0.043) and induced more ISK/HSP70 cells invaded through the filters than mock-infected cells before and after irradiation (274 +/- 21 vs 194 +/- 16 before irradiation, and 298 +/- 24 vs 227 +/- 19 after irradiation, respectively, P = 0.032). CONCLUSION: Disruption of HSP70-induced cytoprotection during irradiation enhances therapeutic effect of irradiation, which makes HSP70 a promising target in the research of endometrial cancer.
Asunto(s)
Línea Celular Tumoral/efectos de la radiación , Neoplasias Endometriales/radioterapia , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Proteínas HSP70 de Choque Térmico/metabolismo , Análisis de Varianza , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral/metabolismo , Supervivencia Celular/efectos de la radiación , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Femenino , Citometría de Flujo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Probabilidad , ARN Mensajero/análisis , Dosis de Radiación , Tolerancia a Radiación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , TransfecciónRESUMEN
The alterations in the gene expression profile of tumor-associated human endometrial endothelial cells (HEECs) may allow opportunities for developing new therapeutic approaches to inhibit angiogenesis in endometrial cancer. The aim of this study was to identify the different gene expression pattern between tumor-associated HEECs and normal HEECs. To elucidate the molecular mechanisms governing the abnormal vasculature in endometrial cancer, we examined global expression patterns of purified endothelial cells from three endometrial cancers and three age-matched normal endometria using oligonucleotide microarrays. We also performed in vitro culture and identified the endothelial origin, as well as observing the functional characteristics in angiogenesis, of HEECs from the two different sources. Microarray analyses revealed distinct gene expression patterns and consistent up-regulation of certain endometrial endothelial marker genes across patient samples. More than 300 genes that exhibited > or =2-fold differences were identified in tumor-associated HEECs. Pathway analysis showed that pathways of Cell cycle, Cell adhesion molecules (CAMs), focal adhesion, and extracellular matrix (ECM)-receptor interaction were obviously predominant. The results of the microarray analysis were confirmed by quantitative real-time PCR, immunohistochemistry, and/or Western blotting. Moreover, although the tumor-associated HEECs did not show faster proliferation than normal HEECs, they exhibited enhanced migration ability, potent invasiveness, and elevated tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, and additional characterization of this gene expression database will provide insights into the angiogenesis of endometrial cancers and might be of great benefit for finding potential therapeutic targets.
Asunto(s)
Neoplasias Endometriales/metabolismo , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica/metabolismo , Movimiento Celular , Neoplasias Endometriales/patología , Células Endoteliales/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Invasividad Neoplásica , Neovascularización Patológica/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Rosiglitazone is known as the most potent and specific peroxisome proliferators-activated receptor gamma (PPAR-gamma) ligand. It has potentially far-reaching effects on pathophysiological processes, from cancer to atherosclerosis and diabetes. However, it is not clear whether rosiglitazone affects the protein expression of transforming growth factor beta3 (TGF-beta3) and the cell proliferation in human uterine leiomyoma cells in vitro. METHODS: Human uterine leiomyoma tissues were dissected and cultured. Cells were divided into 5 groups: one control group and other four groups with different concentrations of rosiglitazone (10(-7), 10(-8), 10(-9) and 10(-10) mol/L). Cells were cultured for 72 hours in serum-free Dulbecco's modified Eagle's medium. MTT reduction assay was used to detect the cell proliferation. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of PPAR-gamma and TGF-beta3. Immunofluorescence staining was used to detect the expressions of PPAR-gamma and TGF-beta3 proteins. RESULTS: MTT reduction assay indicated that the treatment with rosiglitazone (from 10(-7) to 10(-9) mol/L) resulted in an inhibition of the cell growths after 72 hours (P < 0.01). RT-PCR analysis revealed that 10(-7) mol/L rosiglitazone significantly affected the gene expression at 72-hour: PPAR-gamma mRNA expression was up-regulated and TGF-beta3 mRNA was down-regulated and rosiglitazone at the concentration of 10(-7) mol/L affected these most effectively (P < 0.01). Immunofluorescence staining demonstrated that treatment with 10(-7) mol/L rosiglitazone resulted in the significant changes of PPAR-gamma and TGF-beta3 protein expressions compared with the other treatment groups and the control group at 72-hour (P < 0.01). All the effects of rosiglitazone on uterine leiomyoma cells were dose- and time-dependent in vitro. CONCLUSIONS: The present study demonstrates that the PPAR-gamma activator, rosiglitazone, inhibits the cell proliferation partly through the regulations of PPAR-gamma and TGF-beta3 expressions. The cross-talk between the signal pathways of PPAR-gamma and TGF-beta3 may be involved in the process.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leiomioma/tratamiento farmacológico , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Factor de Crecimiento Transformador beta3/genética , Neoplasias Uterinas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Leiomioma/patología , PPAR gamma/análisis , PPAR gamma/genética , ARN Mensajero/análisis , Rosiglitazona , Factor de Crecimiento Transformador beta3/análisis , Neoplasias Uterinas/patologíaRESUMEN
AIM: To explore the role of lysophosphatidic acid receptor-2 (LPA2) in regulating lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) activation, cell invasion, and migration in human ovarian cancer cell line SKOV-3. METHODS: SKOV-3 cells were stimulated with LPA. Cell supernatant uPA level and activity were measured using enzyme-linked immunosorbent assay. LPA2 mRNA expression was inhibited with LPA2-specific small interfering RNA (siRNA) and examined using semiquantitative reverse transcriptase-polymerase chain reaction. LPA-induced cell invasion and migration in transfected cells were evaluated by a Matrigel invasion chamber and a Transwell chemotaxis chamber, respectively. RESULTS: LPA stimulation significantly enhanced in vitro uPA activity in time- and dose-dependent manner. The levels of LPA-induced uPA protein decreased by 55% in LPA2 siRNA-transfected cells compared with negatively transfected cells at 24 hours after being treated with 80 micromol/L LPA (0.75+/-0.03 vs 0.34+/-0.04, P=0.004). In the LPA2 specific siRNA-transfected SKOV-3 cells, LPA treatment at 80 micromol/L induced considerably less invasion and migration compared with negative control siRNA-transfected SKOV-3 cells (invasion: 178+/-17.2 vs 36.2+/-3.3, P=0.009; migration: 220.4+/-25.5 vs 57+/-7.6, P=0.009). CONCLUSION: LPA2 has an essential role in LPA-induced uPA activation and tumor cell invasion in ovarian cancer SKOV-3 cells.
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Lisofosfolípidos/farmacología , Neoplasias Ováricas/fisiopatología , Interferencia de ARN , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Procesos de Crecimiento Celular , Línea Celular Tumoral , Movimiento Celular , Quimiotaxis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas In Vitro , Células K562 , Proyectos Piloto , ARN Mensajero , Transducción de SeñalRESUMEN
Human hematopoietic stem cells (HSCs) are difficult to transfect with retroviral vectors because of their quiescent nature. Based on the theory that the murine fetal stromal cell line AFT024 can recruit significant numbers of HSC into cell cycle without loss of their primitive function, we transduced human umbilical cord blood cells (UCB) derived CD34+ cells with a retroviral vector pHaMDR1/A containing the human multidrug resistant 1 gene (MDR1) during co-culture with the AFT024 feeders. We found that the presence of the AFT024 cells increased the proportion of Rh-123dull cells up to 35.5%+/-11.4% and transduced colony-forming cells (CFCs) up to 15.2%. Six weeks after transplantation of 5x10(4) day 0 uncultured CD34+ HSCs or their equivalents expanded in the presence or absence of the AFT024 cells for 21 days into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, we found that CD34+ cells expanded in the presence of the AFT024 cells engrafted in each receptor mouse and the percentage of CD45+ cells reached 18.8%+/-9.5%, of which 18.1%+/-6.0% were Rh-123dull cells. These results suggest that the AFT024 stromal cells can significantly improve MDR1 gene transfer efficiency and maintain the engrafting ability of the CD34+ HSCs derived from UCB.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antígenos CD4/análisis , Sangre Fetal/citología , Células del Estroma/citología , Transfección/métodos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Animales , Transporte Biológico/fisiología , Línea Celular , Técnicas de Cocultivo , Sangre Fetal/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células 3T3 NIH , Rodamina 123/metabolismo , Trasplante HeterólogoRESUMEN
BACKGROUND: To evaluate the efficacy and safety of 10 mg and 25âmg mifepristone per day compared with 3.75âmg enantone in treating uterine fibroids. METHODS: This is a Multicenter randomized controlled trial. A total of 501 subjects with symptomatic uterine fibroids were enrolled and randomized into the group of 10mg, 25mg mifepristone and 3.75 enantone (with 307, 102 and 92 subjects respectively), with 458 subjects completed the treatment. Three months of daily therapy with oral mifepristone (at a dose of either 10âmg or 25âmg) or once-monthly subcutaneous injections of enantone (at a dose of 3.75 mg) were used. Change in volume of the largest uterine fibroid was the primary efficacy variable, and secondary efficacy variables included changes in anemia and relevant symptom. Safety evaluation included the analyses of adverse events, laboratory values, and relevant endometrial changes. RESULTS: After three months of treatment, the mean volume of the largest leiomyoma was significantly reduced by mifepristone 10 mg or 25 mg or enantone 3.75 mg (40.27%, 42.59% and 44.49% respectively) (Pâ<â0.0001). Percentage change from baseline in largest leiomyoma volume was not statistically significant among the three groups (Pâ=â0.1057). Most of the patients in all groups experienced amenorrhea after the treatment. There were also significant elevations in red blood cell count, hemoglobin and hematocrit (Pâ<â0.0001), and significant reductions in prevalence of dysmenorrhea, pelvic pressure, non-menstrual abdominal pain (Pâ<â0.0001) in each group, while no significant difference among the three groups.All study medications are well-tolerated, and no serious adverse event was reported. Treatment-related adverse event rate was significantly lower in mifepristone 10 mg group, compared to Enantone 3.75 mg group (13.59% vs. 32.58%, Pâ=â0.0002). In both mifepristone groups, estradiol levels were maintained in the premenopausal range, whereas patients in the enantone group had a significant reduction to postmenopausal levels (Pâ<â0.0001). CONCLUSION: 10mg is as effective as 25mg mifepristone and 3.75 mg enantone with minimal drug-related side effects, and may provide an alternative for clinical application, especially for patient who are in perimenopause with uterine fibroids.
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Antineoplásicos Hormonales/uso terapéutico , Antagonistas de Hormonas/uso terapéutico , Leiomioma/tratamiento farmacológico , Leuprolida/uso terapéutico , Mifepristona/uso terapéutico , Adulto , Amenorrea/inducido químicamente , Antineoplásicos Hormonales/efectos adversos , Relación Dosis-Respuesta a Droga , Dismenorrea/tratamiento farmacológico , Femenino , Pruebas Hematológicas , Antagonistas de Hormonas/administración & dosificación , Antagonistas de Hormonas/efectos adversos , Humanos , Leuprolida/efectos adversos , Persona de Mediana Edad , Mifepristona/administración & dosificación , Mifepristona/efectos adversosRESUMEN
OBJECTIVE: To investigate the expression of CD56 in endometrial samples from patients with adenomyosis and its relationship with menstrual cycle phase and severity of dysmenorrhea. STUDY DESIGN: 40 patients with histologically proved adenomyosis (proliferative n=20; secretory n=20) and dysmenorrhea were examined in this study, control groups includes 20 patients with adenomyosis without dysmenorrhea (main complaint: menorrhagia) and 20 patients without adenomyosis who had undergone hysterectomy for non-endometrial pathology (no dysmenorrhea medical history). Immunohistochemical staining against CD56 was performed for the eutopic and ectopic endometrium from patients with adenomyosis and the control samples. The expression of CD56 was determined by calculating the H-score and the severity of dysmenorrhea was determined using the visual analogue scale. The menstrual cycle status and the disease severity were compared to the levels of staining. RESULT(S): CD56 was expressed mainly in the endometrial glandular epithelium in patients with adenomyosis and normal endometrium. The epithelial staining intensity of CD56 in ectopic lesions of adenomyosis with dysmenorrhea was obviously higher than in the corresponding eutopic endometrium and control groups (P<0.01). There were no statistical differences in the expression between normal endometrium, eutopic endometrium of adenomyosis with dysmenorrhea and adenomyostic samples without dysmenorrhea. For eutopic endometrium in adenomyosis with dysmenorrhea, expression was higher in the secretory phases than in the proliferative phase (P<0.05). The increased CD56 immunoreactivity correlated with the severity of dysmenorrhea (spearman rho=0.84, P<0.01). CONCLUSION(S): These findings suggest that the expression of CD56 in adenomyosis is positively associated with the severity of dysmenorrhea. Endometrial glandular epithelium is likely to secrete more CD56 and stimulating nerve growth in the stroma, which could then play a role in the pathogenesis of adenomoysis-related dysmenorrhea.
Asunto(s)
Adenomiosis/metabolismo , Antígeno CD56/análisis , Dismenorrea/etiología , Adenomiosis/complicaciones , Adulto , Endometrio/química , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana EdadRESUMEN
OBJECTIVE: To investigate the effect of high dose mifepristone and high dose progesterone in the treatment of patients with endometrial carcinoma and to explore the possible mechanisms associating with them. METHODS: Thirty untreated patients diagnosed as endometrial carcinoma through dilation and curettage of the uteri were divided into 3 groups at random. Each group was given medroxyprogesterone acetate (MPA), (500 mg/day) or mifepristone (MIF), (100 mg/day) or MIF (100 mg/day) + MPA (500 mg/day) for 5 days respectively. On the sixth day, hysterectomy was performed on these patients. The endometrial cancer specimen of post-hysterectomy was compared with the one of pre-administrating. The morphologic changes of the endometrial cancer cells were observed through light microscope. Immunohistochemistry assay (SP method) was applied to determine the localization and immunoreactive intensity of proliferating cell nuclear antigen (PCNA), estrogen receptor (ER), progesterone receptor (PR), B-cell leukemia lymphoma-2 (bcl-2), bcl-2 associated X protein (bax) and CD(44v6). RESULTS: Better differentiation degree and active excretion were observed in all of the post-hysterectomy endometrial specimen. In the same time, apoptosis of carcinoma cells was observed. The most significant changes were seen in the MIF + MPA group. In the MPA group, the pre-treatment and post-treatment expression of PR (2.9 +/- 1.1, 1.6 +/- 0.8), ER (2.8 +/- 0.9, 1.4 +/- 0.9), PCNA (0.84 +/- 0.10, 0.60 +/- 0.12), bcl-2 (0.236 +/- 0.089, 0.157 +/- 0.981) and CD(44v6) (4.6 +/- 1.8, 2.5 +/- 1.9) were all decreased (all P < 0.01); the expression of bax (0.20 +/- 0.10, 0.42 +/- 0.07) was increased (P < 0.01). In the MIF group, the expression of PR (3.4 +/- 1.0, 1.9 +/- 0.8), ER (2.7 +/- 0.9, 1.2 +/- 0.7), PCNA (0.80 +/- 0.15, 0.65 +/- 0.10), bcl-2 (0.214 +/- 0.097, 0.121 +/- 0.073) were all decreased (all P < 0.01); the expression of bax (0.21 +/- 0.05, 0.44 +/- 0.09) was increased (P < 0.01); no significant change in the expression of CD(44v6) (4.2 +/- 2.0, 4.3 +/- 1.7) was seen (P > 0.05). In the MIF + MPA group, the expression of PR (3.2 +/- 1.0, 0.8 +/- 0.8), ER (2.7 +/- 0.9, 0.7 +/- 0.9), PCNA (0.81 +/- 0.09, 0.25 +/- 0.09), bcl-2 (0.225 +/- 0.091, 0.066 +/- 0.009) and CD(44v6) (4.5 +/- 1.9, 2.7 +/- 1.6) were all decreased (all P < 0.01); the expression of bax (0.22 +/- 0.06, 0.59 +/- 0.09) was increased (P < 0.01); there were significant different expression of PCNA, ER, PR, bax and bcl-2 as compared with the MIF group and the MPA group, respectively (all P < 0.01). The expression of CD(44v6) was significantly different (P < 0.01) between the MIF + MPA group, and the MIF group, but not significantly different between the MIF + MPA group and the MPA group. CONCLUSIONS: The study indicates that high dose progesterone could inhibit the growth, promote apoptosis and inhibit metastasis of endometrial carcinoma, MIF could inhibit the growth and promote apoptosis, MIF + MPA could more strongly inhibit the growth, promote apoptosis and inhibit metastasis of endometrial carcinoma than MIF or MPA, and synergistic effect was observed on the expression of PCNA, ER, PR, bax and bcl-2.
Asunto(s)
Neoplasias Endometriales/tratamiento farmacológico , Mifepristona/administración & dosificación , Progesterona/administración & dosificación , Adulto , Anciano , Quimioterapia Combinada , Neoplasias Endometriales/química , Neoplasias Endometriales/patología , Femenino , Glicoproteínas/análisis , Humanos , Receptores de Hialuranos/análisis , Persona de Mediana Edad , Antígeno Nuclear de Célula en Proliferación/análisis , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisisRESUMEN
Phosphatase and tensin homolog (PTEN) and protein phosphatase type 2A (PP2A) are negative modulators of PI3K/AKT/survivin signaling. To evaluate immunoexpression of PTEN, PP2A and survivin in adenomyosis, ectopic lesions from 28 patients with adenomyosis and endometria from 30 controls without adenomyosis were employed in the study. The expression of PTEN, PP2A and survivin was examined with the use of immunohistochemistry. We found a decreased expression of PP2A and PTEN in adenomyosis. The expression of PTEN showed great individual differences in adenomyosis, although expression of both PP2A and PTEN was lower in adenomyosis than in normal endometria. In contrast, the expression of survivin was higher in adenomyosis. Our results suggest the important role of the PI3K cascade in the pathogenesis and development of adenomyosis.
Asunto(s)
Adenomiosis/metabolismo , Regulación hacia Abajo , Endometrio/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteína Fosfatasa 2/metabolismo , Regulación hacia Arriba , Adenomiosis/patología , Adulto , Algoritmos , Endometrio/citología , Endometrio/patología , Femenino , Humanos , Inmunohistoquímica , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , SurvivinRESUMEN
Preeclampsia, characterized by hypertension and proteinuria, remains a leading cause of maternal morbidity and mortality. Recently, a genome-wide association study (GWAS) identified the single-nucleotide polymorphism, rs2681472, as a new hypertension susceptibility genetic variant. The purpose of this study was to evaluate the association between preeclampsia and rs268172 in a Northern Han Chinese population. We genotyped 1218 unrelated Northern Han Chinese women, including 515 patients with preeclampsia and 703 healthy controls. No significant differences were detected in the allele frequencies between patients and controls (P = .23). When patients were divided into early-onset and late-onset preeclampsia according to gestational age of disease onset, the allele frequencies significantly differed between controls and patients with early-onset preeclampsia (P = .02). Genotype frequencies also were significantly different between controls and patients early-onset preeclampsia when data were analyzed under additive (P = .03) and dominant (P = .009) models. We replicated this association in an independent Northern Han Chinese population and observed a significant difference in the allele frequencies between patients with early-onset preeclampsia and controls (P = .011). We report that rs2681472 is associated with early-onset preeclampsia in Northern Han Chinese women.
Asunto(s)
Pueblo Asiatico/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Polimorfismo de Nucleótido Simple , Preeclampsia/genética , Adulto , Estudios de Casos y Controles , China/epidemiología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Preeclampsia/diagnóstico , Preeclampsia/etnología , Embarazo , Factores de Riesgo , Adulto JovenRESUMEN
OBJECTIVE: To investigate the expression and potential roles of interleukin-10 receptor 1 (IL-10R1) and interleukin-10 receptor 2 (IL-10R2) in adenomyosis. STUDY DESIGN: This prospective study examined 33 women with histologically proven adenomyosis and 21 women without adenomyosis who had undergone hysterectomy for non-endometrial pathology. Comparative immunohistochemistry was used to evaluate the expression and localization of IL-10R1 and IL-10R2. Tissue sections were immunostained with goat anti-human interleukin-10 receptor alpha and rabbit anti-human interleukin-10 receptor beta antibodies. The presence and localization of IL-10R1 and IL-10R2 were evaluated microscopically throughout the menstrual cycle in eutopic and ectopic endometrial tissues of women with adenomyosis, and the results were compared with those for normal endometrium. RESULTS: IL-10R1 and IL-10R2 were mainly expressed by epithelial cells in both women with adenomyosis and controls. Epithelial expression of IL-10R1 and IL-10R2 was higher in adenomyotic samples than in eutopic endometrium of women with adenomyosis or normal endometrium. Moreover, epithelial expression of IL-10R1 was higher in eutopic endometrium of women with adenomyosis than in normal endometrium. Epithelial expression of IL-10R1 showed cyclic variation in eutopic endometrium of women with adenomyosis and normal endometrium, with elevated expression in secretory-phase tissues compared with proliferative-phase tissues. CONCLUSIONS: Intrinsic abnormalities concerning IL-10 and IL-10 receptors may be present in eutopic and ectopic endometria of women with adenomyosis. These findings suggest that IL-10 receptors may be involved in the immunotolerant and/or anti-inflammatory process of adenomyosis.
Asunto(s)
Endometriosis/metabolismo , Endometrio/metabolismo , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Subunidad beta del Receptor de Interleucina-10/metabolismo , Enfermedades Uterinas/metabolismo , Adulto , Análisis de Varianza , Femenino , Humanos , Ciclo Menstrual/metabolismo , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND: Ovarian cancers are often at an advanced stage at diagnosis because early detection is difficult. The poor prognosis of ovarian cancers highlights the crucial need to develop better therapeutic agents and strategies. The objective of this study was to investigate the inhibitory effects of a new modified antisense oligonucleotides targeting vascular endothelial growth factor A (VEGF-A) in SKOV3 ovarian cancer cells. METHODS: Antisense oligonucleotides targeting VEGF-A was designed, synthesized and transfected into SKOV3 ovarian cancer cells. Western blotting and real-time RT-PCR were used to analyze the inhibitory effects of antisense oligonucleotides on VEGF-A protein and mRNA expression. Transwell matrix assay was used to detect cell migration inhibition. RESULTS: The antisense oligonucleotides targeting VEGF-A significantly decreased VEGF-A protein and mRNA expression and inhibited cell migration in SKOV3 ovarian cancer cells. CONCLUSIONS: This new modified antisense oligonucleotides targeting VEGF-A can decrease VEGF-A expression and inhibit cell migration in SKOV3 ovarian cancer cells. This new oligonucleotides may be a promising therapeutic agent for ovarian cancers.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Neoplasias Ováricas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The aim of our study was to evaluate the effects of 5-aza-2'-deoxycytidine (5-azadC) on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of hMLH1 and DNMT3B in human endometrial cancer cell lines. Ishikawa, HHUA, and KLE cell lines were used. After treatment with 5-azadC, cells were measured by MTT to detect the growth inhibition. Flow cytometry analysis was used to evaluate the cell cycle distribution and apoptosis effect. The expression of hMLH1 and DNMT3B was performed by real-time PCR and Western blotting analysis. The methylation status of the hMLH1 gene was monitored by methylation-specific PCR. We confirmed that 5-azadC treatment resulted in growth inhibition, G(2) arrest, and cell apoptosis in human endometrial cancer cell lines. Furthermore, the data obtained by real-time PCR and Western blotting analysis demonstrated that the expression of hMLH1 was up-regulated by 5-azadC treatment in Ishikawa cells, accompanied by down-regulation of DNMT3B expression, when 5-azadC led to cell inhibition, G(2)/M arrest, and apoptosis. Our results suggested that 5-azadC is a potent inhibitor of DNA methyltransferase 3B and induces apoptosis in Ishikawa cells with the up-regulation of hMLH1.