RESUMEN

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Chalcona/farmacología , Chalconas/farmacología , Células 3T3 , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Chalcona/administración & dosificación , Chalconas/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , L-Lactato Deshidrogenasa , Células MCF-7 , Ratones , Estructura Molecular , Regiones Promotoras Genéticas

RESUMEN

AIM: Over 40% of patients with pre-eclampsia are nonresponsive to antihypertensive therapy, but the underlying mechanisms are unknown. We examined the effects of plasma from nonresponsive and responsive patients on endothelial gene expression. PATIENTS & METHODS: PCR array was performed in human umbilical vein endothelial cells (HUVEC) incubated with plasma from nonresponsive (n = 4) and responsive (n = 4) patients. Gene networks and interactions with antihypertensive drugs used in pre-eclampsia were identified by Ingenuity Pathway Analysis. RESULTS: Nifedipine and hydralazine act by upregulate or downregulate genes found to be downregulated or upregulated in HUVEC incubated with plasma from nonresponsive patients. CONCLUSION: Our novel findings suggest that plasma from nonresponsive and responsive patients evoke different responses in HUVEC, and might advance the pharmacogenomics research in pre-eclampsia.

Asunto(s)

Endotelio Vascular/fisiología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , Plasma/fisiología , Preeclampsia/genética , Preeclampsia/terapia , Adulto , Antihipertensivos/farmacología , Antihipertensivos/uso terapéutico , Endotelio Vascular/efectos de los fármacos , Femenino , Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Preeclampsia/diagnóstico , Embarazo

RESUMEN

Although the role of soluble fms-like tyrosine kinase 1 (sFLT-1) in preeclampsia is well-established, the mechanism related to its synthesis remains poorly understood. We evaluated the association among the circulating microRNAs (miRs) and sFLT-1 levels in preeclampsia pregnant women. For this purpose, we measured the plasma sFLT-1 levels in 24 preeclampsia women and selected from these, three subjects with the lowest and three with the highest levels of sFLT-1 in order to screen for potential miRs associated with plasmatic sFLT-1 concentrations using a polymerase chain reaction-array (PCR-array) methodology. From screening results, we found three statistically different expressed miRs with fold change (FC-high/low levels) ≥3.0: miR-195-5p (FC = 5.2 increase in group with high sFLT-1 levels), miR-16-5p (FC = 3.2; increase in group with high sFLT-1 levels), and miR-375 (FC = -3.0; decrease in group with high sFLT-1 levels) which were later validated in all samples (n = 24). To correlate these miRs and plasma sFLT-1 levels, we used two extremes of analysis. In one part, we compared 12 preeclampsia women with the lowest sFLT-1 levels (L-50% group) to 12 with the highest levels (50% H group); and in the second analysis, 6 preeclampsia women (L-25%) from the L group to 6 preeclampsia women from the H group (H-25%). Our results showed increased expression of miR-195-5p in the H group, considering both the analyses with 50%, FC = 2.1 and 25%, FC = 4.2. Regarding other miRs, lack of correlation was found in both analyses (50% and 25%). In conclusion, our data demonstrate an association of higher levels of sFLT-1 with increased expression of miR-195-5p in preeclampsia pregnant women.

Asunto(s)

MicroARNs/sangre , Preeclampsia/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Adulto , Femenino , Humanos , Embarazo , Adulto Joven

RESUMEN

SCOPE: We have previously shown an increase in adipocyte size and lipid content in retroperitoneal white adipose tissue (rWAT) induced by an 8-week high-sugar diet (HSD). In this study, we assessed the effect of a HSD on the transcriptional activity of adipogenic genes in a time-course study to provide insight regarding the genetic networks involved in the rWAT response to dietary sugar. METHODS AND RESULTS: Weaned male Wistar rats were fed a standard chow diet or HSD (68% carbohydrates) for 4, 8 or 12 weeks, and rWAT was removed for histopathology and PCR array (adipogenesis) analyses. The HSD induced adipocyte hypertrophy and hyperplasia in rWAT after 12 weeks of ingestion. Additionally, the HSD altered serum VLDL-cholesterol, triacylglycerol and glucometabolic parameters. Hierarchical clustering revealed HSD-induced changes in the expression patterns of the tested gene set. Pathway analysis, which used the enrichment analysis algorithm of the Thompson Reuters MetaCore platform, associated a cluster of differentially expressed genes with canonical pathways related to regulating adipocyte differentiation and proliferation (p-value < 10(-7)). CONCLUSION: HSD feeding post-weaning increased both the adipocyte size and number by simultaneously up-regulating pro-adipogenic signals (the PPARγ pathway) and down-regulating anti-adipogenic signals (Wnt pathway) in young adults.

Asunto(s)

Adipocitos/efectos de los fármacos , Sacarosa en la Dieta/administración & dosificación , Grasa Intraabdominal/patología , Adipogénesis/efectos de los fármacos , Animales , Composición Corporal/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Aumento de la Célula , Proliferación Celular/efectos de los fármacos , VLDL-Colesterol/sangre , Sacarosa en la Dieta/efectos adversos , Regulación hacia Abajo , Hiperplasia/metabolismo , Hipertrofia/metabolismo , Grasa Intraabdominal/efectos de los fármacos , Grasa Intraabdominal/metabolismo , Masculino , PPAR gamma/genética , PPAR gamma/metabolismo , Ratas , Ratas Wistar , Triglicéridos/sangre , Regulación hacia Arriba , Vía de Señalización Wnt

RESUMEN

AIMS: Hyperglycemia leads to increased production of reactive oxygen species (ROS), which reduces cellular antioxidant defenses and induces several DNA lesions. We investigated the effects on DNA damage of a seven-day hospitalization period in patients with type 2 diabetes mellitus (T2DM) to achieve adequate blood glucose levels through dietary intervention and medication treatment, compared with non-diabetic individuals. METHODS: DNA damage levels were evaluated by the alkaline comet assay (with modified and without conventional use of hOGG1 enzyme, which detects oxidized DNA bases) for 10 patients and 16 controls. Real time PCR array method was performed to analyze the transcriptional expression of a set of 84 genes implicated in antioxidant defense and response to oxidative stress in blood samples from T2DM patients (n=6) collected before and after the hospitalization period. RESULTS: The seven-day period was sufficient to improve glycemic control and to significantly decrease (p<0.05) DNA damage levels in T2DM patients, although those levels were slightly higher than those in control subjects. We also found a tendency towards a decrease in the levels of oxidative DNA damage in T2DM patients after the hospitalization period. However, for all genes analyzed, a statistically significant difference in the transcriptional expression levels was not observed. CONCLUSIONS: The study demonstrated that although the transcriptional expression of the genes studied did not show significant alterations, one-week of glycemic control in hospital resulted in a significant reduction in DNA damage levels detected in T2DM patients, highlighting the importance of an adequate glycemic control.

Asunto(s)

Glucemia/análisis , Daño del ADN , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/terapia , Hiperglucemia/terapia , Estrés Oxidativo/genética , Adulto , Femenino , Perfilación de la Expresión Génica , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Especies Reactivas de Oxígeno/sangre

RESUMEN

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-i, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.

Asunto(s)

Humanos , Enterovirus Humano A/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Rabdomiosarcoma/virología , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Enterovirus Humano A/enzimología , Enterovirus Humano A/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/fisiología , Reacción en Cadena de la Polimerasa , Rabdomiosarcoma/enzimología , Rabdomiosarcoma/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba , Replicación Viral

RESUMEN

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8h and 20h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p<0.05). At 8h after infection, the expressions of c-Jun, c-Fos, IFN-ß, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.

Asunto(s)

Enterovirus Humano A/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Rabdomiosarcoma/virología , Citocinas/genética , Enterovirus Humano A/enzimología , Enterovirus Humano A/fisiología , Ensayo de Inmunoadsorción Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Reacción en Cadena de la Polimerasa , Rabdomiosarcoma/enzimología , Rabdomiosarcoma/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba , Replicación Viral