RESUMEN
We theoretically and experimentally investigate double-electromagnetically-induced transparency (double-EIT) cooling of two-dimensional ion crystals confined in a Paul trap. The double-EIT ground-state cooling is observed for ^{171}Yb^{+} ions with a clock state, for which EIT cooling has not been realized like many other ions with a simple Λ scheme. A cooling rate of n[over ¯][over Ë]=34(±1.8) ms^{-1} and a cooling limit of n[over ¯]=0.06(±0.059) are observed for a single ion. The measured cooling rate and limit are consistent with theoretical predictions. We apply double-EIT cooling to the transverse modes of two-dimensional (2D) crystals with up to 12 ions. In our 2D crystals, the micromotion and the transverse mode directions are perpendicular, which makes them decoupled. Therefore, the cooling on transverse modes is not disturbed by micromotion, which is confirmed in our experiment. For the center of mass mode of a 12-ion crystal, we observe a cooling rate and a cooling limit that are consistent with those of a single ion, including heating rates proportional to the number of ions. This method can be extended to other hyperfine qubits, and near ground-state cooling of stationary 2D crystals with large numbers of ions may advance the field of quantum information sciences.
RESUMEN
Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus-infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Hedgehog/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Receptor Smoothened/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Apoptosis/efectos de los fármacos , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Neoplasias Ováricas/metabolismo , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Recently, a growing body of evidence has suggested that abnormal ovarian angiogenesis, secondary to the imbalance between various angiogenic markers, is involved in the pathogenesis of PCOS, and this has led to the use of various interventions (such as Diane-35) to restore the normal ovarian angiogenesis. Therefore, we conducted the current investigation to determine the role of such markers (endothelial growth factor (VEGF), endostatin (ES), and thrombospondin-1 (TSP-1)) in the pathogenesis of PCOS along with the associated changes in ovarian blood flow in patients with PCOS compared to healthy controls, both before and after a course of oral contraception. A total of 381 patients with PCOS and 98 healthy females of childbearing age were recruited from July 2014 to June 2017 at the Reproductive Center of the Second Affiliated Hospital of Harbin Medical University. The serum levels of VEGF, ES, and TSP-1 were determined by enzyme-linked immunosorbent assay, while ovarian perfusion was measured by the pulsatility index (PI) and resistance index (RI) by using transvaginal color Doppler ultrasound. Repeated analyses were carried out after 3 months of Diane-35 treatment. Post-treatment serum levels of luteinizing hormone (LH)/follicle stimulating hormone (FSH) ratio of patients with PCOS decreased significantly (P <0.05). The RI values of most PCOS patients increased after treatment (P<0.05), while PI was significantly increased in all patients (P<0.05). However, variable changes in the serum levels of TSP-1, VEGF, and ES after treatment were observed. Serum VEGF levels showed a negative correlation with serum LH/FSH ratio, T concentration, and ES (P <0.05), while ES levels were negatively correlated with serum T concentrations only (P<0.05). The markers of angiogenesis (VEGF, ES, and TSP-1) were expressed differently among PCOS patients, who also responded differently to the same course of Diane-35 treatment. This field still warrants further investigation to reach a more definitive conclusion.
Asunto(s)
Agentes Anticonceptivos Hormonales/uso terapéutico , Endostatinas/sangre , Síndrome del Ovario Poliquístico/sangre , Trombospondina 1/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Adulto , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Hormona Luteinizante/sangre , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Resultado del Tratamiento , Adulto JovenRESUMEN
Gastric cancer (GC) is one of the most prevalent gastrointestinal malignancies. Long noncoding RNA (lncRNA) DANCR is a newly identified oncogenic lncRNA. However, the functional role and underlying molecular mechanisms of DANCR involved in GC progress remain unclear. In the present study, we investigated the biological function and underlying mechanisms of DANCR in GC cell migration and invasion. The results showed that knockdown of DANCR inhibited migration and invasion of GC cells, whereas overexpression of DANCR showed the opposite effect. Further investigation demonstrated that lncRNA-LET was a bona fide target gene of DANCR. In addition, high DANCR and low lncRNA-LET were significantly correlated with lymph node metastasis and late clinical stage. DANCR associated with EZH2 and HDAC3 to epigenetically silence lncRNA-LET and then regulated GC migration and invasion. Taken together, these findings indicate an important role for DANCR-lncRNA-LET axis in GC cell migration and invasion, and reveal a novel epigenetic mechanism for lncRNA-LET silencing.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , ARN Largo no Codificante/genética , Neoplasias Gástricas/genética , Anciano , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Histona Desacetilasas/metabolismo , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Cisplatin is a first generation platinumbased chemotherapeutic agent, however, the extensive application of cisplatin inevitably results in drug resistance, which is a major obstacle in cancer chemotherapy. The aim of the present study was to investigate the efficiency of reversing cisplatinresistance with the use of combination therapy with oridonin and cisplatin in human ovarian cancer cells, and attempt to reduce the side effects of the therapeutic agents when used alone. The half maximal inhibitory concentration (IC50) values of cisplatin were determined in cisplatinsensitive and cisplatinresistant ovarian cancer cells using an MTT assay. IC50 values of cisplatin in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells were significantly decreased in a timedependent manner. The antitumor effect of oridonin in A2780/DDP cells was also detected by the MTT assay and the inhibitory effects of oridonin increased in a dose and timedependent manner. A2780/DDP cells were treated with 20 µM oridonin in combination with increasing concentrations of cisplatin for 48 h, and the result demonstrated that oridonin synergistically increased the antitumor effects of cisplatin in A2780/DDP cells. Notably, the combination treatment of oridonin and cisplatin effectively reversed cisplatin resistance and the IC50 values were significantly decreased from 50.97 µM and 135.20 to 26.12 µM and 73.00 µM in A2780/DDP and SKOV3/DDP cells at 48 h, respectively. Furthermore, oridonin induced cell apoptosis in a dosedependent manner and promoted cellcycle arrest at the G0/G1 phase in ovarian cancer cells. Oridonin and cisplatin synergistically increased the cell apoptosis rate of A2780/DDP cells, which was detected by fluorescenceactivated cell sorting analysis. Downregulated expression levels of Bcl2 and upregulated the expression of Bax protein were demonstrated by western blot analysis, further indicating increased apoptosis. In addition, the expression levels of matrix metalloproteinase (MMP)2 and MMP9 decreased in a dosedependent manner with oridonin treatment. The results from the present study demonstrated that oridonin exerted a synergistic effect with cisplatin to inhibit proliferation and induce cell apoptosis in cisplatinresistant ovarian cancer cells. Thus, combination therapy with oridonin and cisplatin effectively reversed cisplatin resistance in human ovarian cancer cells, which may have useful clinical applications.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Diterpenos de Tipo Kaurano/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/química , Diterpenos de Tipo Kaurano/química , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The aim of the present study was to examine the effects of the human chorionic gonadotropin (hCG) dose on the pulsatility indices (PI) of the intraovarian artery on the day of follicle aspiration and the oocyte quality, intrafollicular oxidative stress and luteinization. PI was also measured on the day of hCG administration. A total of 15 patients were undergoing the in vitro fertilization and embryo transfer (IVF-ET) program. To estimate whether there was any difference between the intraovarian artery blood flow and oocyte development of the same patients treated with 5,000 or 10,000 IU hCG, the intraovarian artery blood flow was measured by transvaginal color ultrasonography pulsed wave Doppler, and the follicular fluids and the granulosa cells were collected at follicle aspiration. There were statistically significant differences between the same patients undergoing the two different hCG-dose treatments in which the first protocol included 10,000 IU and the second protocol included 5,000 IU hCG treatment. These differences were apparent in the PI of intraovarian artery blood flow on the day of follicle aspiration (P=0.0023), in the incidence of apoptosis in cumulus (ApoC) and mural (ApoM) granulosa cells (ApoC, P=0.0077; ApoM, P=0.0128), in the total oocytes retrieved (P=0.0342) and in the follicle fluid progesterone concentration (P=0.0044). There were no significant differences between the two protocols in the PI of intraovarian artery blood flow on the day of hCG administration (P=0.4326), serum steroid on the day of follicle aspiration [serum P, P>0.9999; serum estradiol (E2), P=0.8589], follicle fluid E2 concentration (P=0.8939), mature oocyte rate (P=0.3743) and total mature oocytes retrieved (P=0.2026). In conclusion, the dose of hCG administration can significantly affect the intraovarian artery blood flow and the development of follicles and oocytes in an IVF-ET program.
RESUMEN
The aim of the present study was to investigate the clinical value of lowdose aspirin in combination with Tiao Jing Cu Yun pills in patients with polycystic ovary syndrome (PCOS) by measuring follicular peripheral blood flow parameters and the clinical efficacy. The study involved 78 infertile females with PCOS who were randomly divided into experimental (n=38) and control (n=40) groups. The subjects in the experimental group were treated with letrozole in combination with aspirin and Tiao Jing Cu Yun pills, and the control group was treated with letrozole alone. Transvaginal color Doppler ultrasonography was used to measure the endometrial thickness, pulsatility index (PI), resistance index (RI), peak systolic velocity and end diastolic velocity (EDV) of the follicular peripheral artery on the day of human chorionic gonadotropin administration. The patients who failed to become pregnant in the control group were reintegrated into the experimental group in the subsequent cycle and the clinical effect was observed. In the experimental group, subject perifollicular blood flow was more plentiful, and the PI and RI of the perifollicular blood flow were significantly reduced, while the EDV of the perifollicular blood flow and the rate of clinical pregnancy were markedly elevated compared with the subjects in the control group. Lowdose aspirin combined with Tiao Jing Cu Yun pills effectively improved perifollicular artery blood flow, and enhanced the oocyte quality and rate of clinical pregnancy.
Asunto(s)
Aspirina/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Fármacos para la Fertilidad Femenina/administración & dosificación , Infertilidad Femenina/tratamiento farmacológico , Folículo Ovárico/irrigación sanguínea , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Adulto , Quimioterapia Combinada , Endometrio/efectos de los fármacos , Endometrio/patología , Femenino , Humanos , Letrozol , Nitrilos/administración & dosificación , Folículo Ovárico/diagnóstico por imagen , Embarazo , Índice de Embarazo , Triazoles/administración & dosificación , Ultrasonografía , Adulto JovenRESUMEN
Estrogen (E2) has direct in vivo and in vitro effects, such as inducing neurite outgrowth, on neurons. We investigated the morphological changes and intracellular signaling pathway induced by E2 in neuroblastoma (SH-SY5Y) cells. The effect of medroxyprogesterone acetate (MPA) or progesterone (P4) on the E2-induced neurite outgrowth was also examined using SH-SY5Y cells. Neurite outgrowth was induced by E2 in association with the phosphorylation of Akt, and these effects of E2 were abolished by MPA but not by P4. Progesterone receptor antagonist RU486 blocked the inhibitory effects of MPA. Estrogen receptor antagonist ICI 182,780 and phosphatidylinositol 3-kinase inhibitor LY294002 inhibited the E2-induced neurite outgrowth. Because the Rho family of small GTPases has been shown to be involved in the regulation of neurite outgrowth, we examined the cross-talk among Rac1, Cdc42 and RhoA in the E2-induced neurite outgrowth. E2 immediately increased the Rac1 and Cdc42 activity and decreased the RhoA activity. E2-induced neurite outgrowth was attenuated in cells expressing dominant-negative mutants for Rac1 or Cdc42. These results suggest that regulation of Rho family GTPase activity by E2 is important for the neurite outgrowth in neuroblastoma cells, and that MPA may have an antagonistic effect against E2.
Asunto(s)
Estradiol/farmacología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Línea Celular Tumoral , Humanos , Acetato de Medroxiprogesterona/farmacología , Mifepristona/farmacología , Neuritas/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Progesterona/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/efectos de los fármacos , Clorhidrato de Raloxifeno/farmacología , Telomerasa/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Humanos , Morfolinas/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
OBJECTIVE: To examine the correlation between the pulsatility and resistance indices (PI and RI) of the intraovarian artery on the day of follicle aspiration and the oocyte quality, intrafollicular oxidative stress, and luteinization. Pulsatility index and RI on the day of hCG administration also were measured. DESIGN: Prospective study. SETTING: Obstetrics and gynecology department of a university medical school in Japan. PATIENT(S): Thirty-five patients in an IVF-ET program. INTERVENTION(S): The PI and RI of the intraovarian artery were measured by transvaginal color ultrasonographic pulsed wave Doppler on the day of hCG administration and the day of follicle aspiration. Follicular fluids and the granulosa cells were collected at follicle aspiration. MAIN OUTCOME MEASURE(S): The PI and RI of the intraovarian artery blood flow on the day of hCG administration and of follicle aspiration, as well as the rate of development of mature oocytes, follicular fluid steroid levels, and the incidence of apoptosis in the granulosa cells. RESULT(S): The PI and RI on the day of follicle aspiration were correlated positively with the rate of mature oocytes retrieved (PI: r = 0.429; RI: r = 0.348), were correlated negatively with the incidence of apoptotic mural (PI: r = -0.383; RI: r = -0.459) and cumulus (PI: r = -0.378; RI: r = -0.469) granulosa cells, and were negatively correlated with the concentration of P in the follicular fluid (PI: r = -0.429; RI: r = -0.359). The PI and RI on the day of hCG administration were negatively correlated only with the total number of retrieved oocytes (PI: r = -0.393; RI: r = -0.374). CONCLUSION(S): The PI and RI of the intraovarian artery blood flow measured on the day of follicle aspiration may be good indicators of the follicle luteinization and oxidation as well as of oocyte quality.
Asunto(s)
Infertilidad/fisiopatología , Folículo Ovárico , Ovario/irrigación sanguínea , Pulso Arterial , Succión , Resistencia Vascular , Adulto , Apoptosis , Arterias/diagnóstico por imagen , Gonadotropina Coriónica/uso terapéutico , Femenino , Líquido Folicular/química , Células de la Granulosa , Humanos , Oocitos , Concentración Osmolar , Progesterona/análisis , Factores de Tiempo , Recolección de Tejidos y Órganos , Ultrasonografía Doppler de PulsoRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-kappaB in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.
Asunto(s)
Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Androstadienos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , FN-kappa B/metabolismo , Neoplasias Ováricas/patología , Fosforilación , Wortmanina , Proteína Letal Asociada a bcl/metabolismoRESUMEN
The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclina D1/metabolismo , Acetato de Medroxiprogesterona/farmacología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Fosforilación/efectos de los fármacos , Regulación hacia ArribaRESUMEN
In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.
Asunto(s)
Neoplasias de la Mama , Factor de Crecimiento Epidérmico/farmacología , Receptor alfa de Estrógeno/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Células COS , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Mutagénesis , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
Although estrogen is known to protect against beta-amyloid (Abeta)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Abeta-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Abeta-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) alpha gene (PCER). Raloxifene, like 17beta-estradiol (E2), significantly inhibited Abeta-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Abeta, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2- and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor kappaB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Estrógenos/farmacología , Enfermedades Neurodegenerativas/prevención & control , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Western Blotting/métodos , Encéfalo/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática , Humanos , Enfermedades Neurodegenerativas/metabolismo , Feocromocitoma , Proteínas Proto-Oncogénicas c-akt , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Transfección/métodosRESUMEN
The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17beta-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFkappaB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFkappaB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFkappaB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFkappaB.