RESUMEN
Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.
Asunto(s)
Autofagia/genética , Proteína Quinasa CDC2/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteína Fosfatasa 2/genética , Factor de Transcripción Sp1/genética , Autofagia/efectos de los fármacos , Western Blotting , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Cantaridina/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Inhibidores Enzimáticos/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células MCF-7 , Ácido Ocadaico/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: To investigate the chemical constituents from the leaves of Vaccinium bracteatum. METHOD: Many column chromatographic techniques were used for the isolation and separation of chemical constituents. Their structures were elucidated on the basis of spectral analysis and chemical evidences. RESULT: Twelve compounds were isolated from the plant, and they were identified as chrysoeriol (1), scopoletin (2), trans-p-hydroxycinnamic acid (3), trans-p-hydroxycinnamic acid ethyl ester (4), cafeic acid ethyl ester (5), beta-sitosterol (6), iuteolin (7), quercetin (8), esculetin (9), cafeic acid (10), isolariciresinol-9-O-beta-D-xyloside (11), 10-O-trans-p-coumaroylsandoside (12). CONCLUSION: Compounds 4, 5, 11, 12 were isolated from the genus Vaccinium for the first time, and compounds 1, 2, 9, 10 were isolated from this plant for the first time.
