RESUMEN
Previously, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29. HT-29 cells have been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis. In this study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in HT-29 cells. We showed that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK pathway activation and subsequent DR5 overexpression. Furthermore, we showed that diosgenin alone, TRAIL alone or combination treatment increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction.
Asunto(s)
Apoptosis/efectos de los fármacos , Diosgenina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Western Blotting , Ciclooxigenasa 2/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Células HT29 , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microscopía de Contraste de Fase , Piridinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Daphne gnidium L. is a well-known Moroccan plant with cancer-related ethnobotanical use. In order to systematically evaluate its potential activity in breast cancer, four extracts from this plant of different polarity were tested for their antiproliferative effects on MCF-7 cells. The second aspect of this study related to understanding the nature and mechanism of the antiproliferative effect. Results from a viability assay showed the potent antiproliferative capacity of the hexane (IC(50)-48 h: 630 +/- 16 microg/ml), dichloromethane (IC(50)-48 h: 112 +/- 7 microg/ml) and ethyl acetate extracts (IC(50)-48 h: 263 +/- 9 microg/ml). On the other hand the methanol extract was inactive. LDH test revealed the cytotoxicity of the hexane extract as opposed to two others. The characterization of the ethyl acetate extract showed its dose-dependent pro-apoptotic effect. Surprisingly, we observed that activation of the inducible cyclooxygenase-2 followed the kinetics of apoptosis development. On the other hand, the dichloromethane extract showed a distinct effect on COX-2 activity as a function of the used dose. A low dose seemed to inhibit COX-2 activity whereas a high dose seemed to increase it. These findings suggest that Daphne gnidium L. might be of potential chemopreventive interest. Other studies are in hand to isolate the active agents responsible for the antiproliferative effect.
Asunto(s)
Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daphne/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas para Inmunoenzimas , L-Lactato Deshidrogenasa/metabolismo , Raíces de Plantas/químicaRESUMEN
Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.
Asunto(s)
Diferenciación Celular , Diosgenina/metabolismo , Leucemia Eritroblástica Aguda/patología , Megacariocitos/patología , Secuencia de Bases , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Fraccionamiento de Campo-Flujo , Humanos , Ploidias , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrofotometría UltravioletaRESUMEN
Rheumatoid arthritis (RA) is characterized by persistent joint synovial tissue inflammation. Leflunomide is an immunomodulatory agent that has been approved for treatment of active RA. In the past few years, uses other than RA treatment have appeared. Leflunomide has been reported to show antitumor potential through inhibition of cancer cell proliferation. We thus tested the antiproliferative potential of leflunomide on HEL and K562 erythroleukemia cells. The findings summarized in this report demonstrate for the first time that low dose leflunomide prolonged survival and reduced apoptosis induced by several anticancer agents in erythroleukemia cells. We showed that in treated cells, leflunomide reduced the signalling pathways involved in promoting apoptosis by reducing p38 MAPK and JNK basal activity. On the other hand, leflunomide transiently activated the ERK signalling pathway and induced a sustained activation of Akt. We also showed that leflunomide reduced caspase-3 activity and DNA fragmentation induced by anticancer agents. By using an inhibitory strategy, we showed that inhibition of Akt activation but not ERK abolished the protective effect of leflunomide. Thus our findings suggested that leflunomide reduced apoptosis induced by anticancer agents through PI3K/Akt signalling activation.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Isoxazoles/farmacología , Leucemia Eritroblástica Aguda/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Etopósido/análogos & derivados , Etopósido/farmacología , Humanos , Células K562 , Leflunamida , Leucemia Eritroblástica Aguda/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Compuestos Organofosforados/farmacología , Estaurosporina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.
Asunto(s)
Apoptosis , Separación Celular/métodos , Diosgenina/farmacología , Fraccionamiento de Campo-Flujo/métodos , Línea Celular Tumoral , Diosgenina/metabolismo , Fraccionamiento de Campo-Flujo/instrumentación , Humanos , Técnicas In VitroRESUMEN
Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.