ABSTRACT
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.
Subject(s)
Autophagy/genetics , CDC2 Protein Kinase/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , G2 Phase Cell Cycle Checkpoints/genetics , JNK Mitogen-Activated Protein Kinases/genetics , Protein Phosphatase 2/genetics , Sp1 Transcription Factor/genetics , Autophagy/drug effects , Blotting, Western , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , Cantharidin/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Down-Regulation/drug effects , Down-Regulation/genetics , Enzyme Inhibitors/pharmacology , G2 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , MCF-7 Cells , Okadaic Acid/pharmacology , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/metabolism , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
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.
